Methods of forming protein-linked lipidic microparticles, and compositions thereof

ABSTRACT

The present invention provides for lipid:nucleic acid complexes that have increased shelf life and high transfection activity in vivo following intravenous injection, and methods of preparing such complexes. The methods generally involve contacting a nucleic acid with an organic polycation to produce a condensed nucleic acid, and then combining the condensed nucleic acid with a lipid comprising an amphiphilic cationic lipid to produce the lipid:nucleic acid complex. This complex can be further stabilized by the addition of a hydrophilic polymer attached to hydrophobic side chains. The complex can also be made specific for specific cells, by incorporating a targeting moiety such as an Fab′ fragment attached to a hydrophilic polymer. The present invention further relates to lipidic microparticles with attached proteins which have been first conjugated to linker molecules having a hydrophilic polymer domain and a hydrophobic domain capable of stable association with the microparticle, or proteins which have been engineered to contain a hydrophilic domain and a lipid moiety permitting stable association with the microparticle.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/967,791,filed Nov. 10, 1997, and of Provisional Application U.S. Ser. No.60/030,578, filed Nov. 12, 1996, both of which are incorporated hereinby reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to the field of cationic lipid: DNAcomplexes (“CLDC”). In particular, the present invention relates tolipid:nucleic acid complexes that contain (1) hydrophilic polymer; (2)nucleic acid that has been condensed with organic polycations; and (3)hydrophilic polymer and nucleic acid that has been condensed withorganic polycations. The lipid:nucleic acid complexes of this inventionshow high transfection activity in vivo following intravenous injectionand an unexpected increase in shelf life, as determined by in vivotransfection activity.

The present invention further relates to the field of lipidicmicroparticles, such as liposomes, lipid:DNA complexes, lipid:drugcomplexes, and microemulsion droplets, attached to proteins. Inparticular, the invention relates to lipidic microparticles withattached proteins which have been first conjugated to linker moleculeshaving a hydrophilic polymer domain and a hydrophobic domain capable ofstable association with the microparticle, or proteins which have beenengineered to contain a hydrophilic domain and a lipid moiety permittingstable association with a lipidic microparticle.

BACKGROUND OF THE INVENTION

Liposomes that consist of amphiphilic cationic molecules are usefulnon-viral vectors for gene delivery in vitro and in vivo (reviewed inCrystal, Science 270: 404-410 (1995); Blaese et al., Cancer Gene Ther.2: 291-297 (1995); Behr et al., Bioconjugate Chem. 5: 382-389 (1994);Remy et al., Bioconjugate Chem. 5: 647-654 (1994); and Gao et al., GeneTherapy 2: 710-722 (1995)). In theory, the positively charged liposomescomplex to negatively charged nucleic acids via electrostaticinteractions to form lipid:nucleic acid complexes. The lipid:nucleicacid complexes have several advantages as gene transfer vectors. Unlikeviral vectors, the lipid:nucleic acid complexes can be used to transferexpression cassettes of essentially unlimited size. Since the complexeslack proteins, they may evoke fewer immunogenic and inflammatoryresponses. Moreover, they cannot replicate or recombine to form aninfectious agent and have low integration frequency.

There are a number of publications that demonstrate convincingly thatamphiphilic cationic lipids can mediate gene delivery in vivo and invitro, by showing detectable expression of a reporter gene in culturecells in vitro (Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-17(1987); Loeffler et al., Methods in Enzymology 217: 599-618 (1993);Felgner et al., J. Biol. Chem. 269: 2550-2561 (1994)). Becauselipid:nucleic acid complexes are on occasion not as efficient as viralvectors for achieving successful gene transfer, much effort has beendevoted in finding cationic lipids with increased transfectionefficiency (Behr, Bioconjugate Chem. 5: 382-389 (1994); Remy et al.,Bioconjugate Chem. 5: 647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995)). Lipid:nucleic acid complexes are regarded withenthusiasm as a potentially useful tool for gene therapy.

Several groups have reported the use of amphiphilic cationiclipid:nucleic acid complexes for in vivo transfection both in animals,and in humans (reviewed in Gao et al., Gene Therapy 2: 710-722 (1995);Zhu et al., Science 261: 209-211 (1993); and Thierry et al., Proc. Natl.Acad. Sci. USA 92: 9742-9746 (1995)). However, the technical problemsfor preparation of complexes that have stable shelf-lives have not beenaddressed. For example, unlike viral vector preparations, lipid:nucleicacid complexes are unstable in terms of particle size (Behr,Bioconjugate Chem. 5: 382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2: 710-722 (1995)). It istherefore difficult to obtain homogeneous lipid:nucleic acid complexeswith a size distribution suitable for systemic injection. Mostpreparations of lipid:nucleic acid complexes are metastable.Consequently, these complexes typically must be used within a shortperiod of time ranging from 30 minutes to a few hours. In recentclinical trials using cationic lipids as a carrier for DNA delivery, thetwo components were mixed at the bed-side and used immediately (Gao etal., Gene Therapy 2: 710-722 (1995)). The structural instability alongwith the loss of transfection activity of lipid:nucleic acid complexwith time have been challenges for the future development oflipid-mediated gene therapy.

Liposomes consisting of amphiphilic cationic molecules are not, ofcourse, the only form of lipidic microparticles and gene therapy is notthe only utility for such particles. Lipidic microparticles have alsobeen used for delivery of drugs and other agents to target sites.Targeting of the microparticles is typically achieved through use of aprotein attached to the surface of the microparticle, which may, forexample be a ligand for cell surface receptor on a cell type ofinterest. Conversely, the protein may be an antibody which specificallyrecognizes an antigen on a cell type of interest, such as diseased cellscarrying specific markers. Additionally, proteins can be attached forpurposes other than targeting. For example, liposomes can containprodrugs which slowly seep from the liposome into the circulation. Anenzyme attached to the liposome can then convert the prodrug into itsactive form.

Current methods for effecting the attachment of proteins to lipidicmicroparticles have been of two types. The first type requiresintroducing a linker molecule bearing an “active” group (one whichreacts with a functional group of the protein) into the microparticlecomposition prior to conjugation of the “activated” particle with theprotein of interest. The disadvantages of methods of this type are:often uncontrollable, incomplete reaction of the protein with thelinker; the presence of excess linker on the resulting conjugate,potentially adverse effect of the linker on the stability of theparticle, and the inability to incorporate components reactive with thelinker into the composition of the particle.

The second group of methods employs the steps of (a) attachment of ahydrophobic moiety, such as a hydrocarbon chain, to the proteinmolecule, (b) dissolving the components of the lipidic microparticle,along with the conjugate of step (a) in the presence of a detergent, and(c) removing the said detergent, effecting the formation of the lipidicparticle incorporating the protein conjugate (Torchilin, Immunomethods4-244-258 (1994); Laukkanen et al., Biochemistry 33:11664-11670 (1994)).These methods have a number of disadvantages, including the impositionof severe limitations on the range of methods by which the particle canbe formed, (e.g. the detergent removal technique is required) and bywhich the drug or other agent can be loaded into the microparticle.Moreover, step (b) requires the dissolution of the microparticle. Thesemethods are therefore unable to attach a protein to a premade particlewithout first destroying it. The presence of detergent in these methodsis unavoidable because without a detergent the hydrophobically modifiedprotein is insoluble in aqueous medium

The “insertion” into liposomes of hydrophilic polymer-lipid linked to asmall (5 amino acid) oligopeptide or small oligosaccharide has beenreported. (Zalipsky et al., Bioconjugate Chem. 8:111-118 (1997). Thepeptide and oligosaccharide employed were, however, of a size (molecularweight, 500-3,000 Da) smaller than, or comparable to, the linker itself(molecular weight 2,750 Da). This study therefore provides no guidancefor inserting into liposomes or other lipidic microparticles proteins,such as antibodies, or fragments thereof, conjugated to linkerssignificantly smaller than the protein. In view of the hydrophilicnature of antibodies and other proteins, the art has taught that thelarger, protein portion of such a conjugate prevents the hydrophobiclinking moiety from stable association with a lipidic microparticle.

SUMMARY OF THE INVENTION

The present invention provides a novel method of preparing cationiclipid:nucleic acid complexes that have increased shelf life. In oneembodiment, these complexes are prepared by contacting a nucleic acidwith an organic polycation, to produce a condensed or partiallycondensed nucleic acid. The condensed nucleic acid is then combined withan amphiphilic cationic lipid plus a neutral helper lipid such ascholesterol in a molar ratio from about 2:1 to about 1:2, producing thelipid:nucleic acid complex. Optionally, a hydrophilic polymer issubsequently added to the lipid:nucleic acid complex. Alternatively, thehydrophilic polymer is added to a lipid:nucleic acid complex comprisingnucleic acid that has not been not condensed. These lipid:nucleic acidcomplexes have an increased shelf life, e.g., when stored at 22° C. orbelow, as compared to an identical lipid:nucleic acid complex in whichthe nucleic acid component has not been contacted with the organicpolycation and/or in which the lipid:nucleic acid complex has not beencontacted with a hydrophilic polymer.

In a particularly preferred embodiment, the polycation is a polyamine,more preferably a polyamine such as spermidine or spermine.

In another preferred embodiment, the lipid:nucleic acid complexes areprepared by combining a nucleic acid with an amphiphilic cationic lipidand then combining the complex thus formed with a hydrophilic polymer.This lipid:nucleic acid complex has an increased shelf life, e.g., whenstored at 22° C. or below as compared to an identical complex that hasnot been combined with the hydrophilic polymer.

In one embodiment, the hydrophilic polymer is selected from the groupconsisting of polyethylene glycol (PEG), polyethylene glycol derivatizedwith phosphatidyl ethanolamine (PEG-PE), polyethylene glycol derivatizedwith tween, polyethylene glycol derivatized withdistearoylphosphatidylethanolamine (PEG-DSPE), ganglioside G_(M1) andsynthetic polymers.

In one embodiment, the lipid:nucleic acid complex is lyophilized.

In any of the methods and compositions of this invention, the nucleicacid can be virtually any nucleic acid, e.g., a deoxyribonucleic acid(DNA) or a ribonucleic acid (RNA), and peptide nucleic acid (PNA) etc.,and is most preferably a DNA. In a particularly preferred embodiment,the DNA is an expression cassette capable of expressing a polypeptide ina cell transfected with the lipid:nucleic acid complex.

In one embodiment the lipid:nucleic acid complexes are formed by firstforming a liposome, and then combining the formed liposome withcondensed or partially condensed nucleic acid to form a lipid:nucleicacid complex. Optionally, the lipid:nucleic acid complex is subsequentlycontacted with a hydrophilic polymer. The liposomes can alternatively becombined with an uncondensed nucleic acid to form a lipid:nucleic acidcomplex to which a hydrophilic polymer (e.g., PEG-PE) is later added. Alipid:nucleic acid complex prepared by the combination of nucleic acidand a liposome contacted with a hydrophilic polymer can be subsequentlycombined with additional hydrophilic polymer. In a preferred embodiment,the lipid and nucleic acid are combined in a ratio ranging from about 1to about 20, more preferably from about 4 to about 16, and mostpreferably from about 8 to about 12 nmole lipid:μg nucleic acid. Thelipid and hydrophilic polymer are combined in a molar ratio ranging fromabout 0.1 to about 10%, more preferably from about 0.3 to about 5% andmost preferably from about 0.5% to about 2.0% (molar ratio ofhydrophilic polymer to cationic lipid of the complex).

It will be appreciated that a targeting moiety (e.g., an antibody or anantibody fragment) can be attached to the lipid and/or liposome beforeor after formation of the lipid:nucleic acid complex. In a preferredembodiment, the targeting moiety is coupled to the hydrophilic polymer(e.g., PEG), where the targeting moiety/hydrophilic polymer issubsequently added to the lipid:nucleic acid complex. This provides aconvenient means for modifying the targeting specificity of an otherwisegeneric lipid:nucleic acid complex.

In a particularly preferred embodiment, the method of increasing theshelf life of the lipid:nucleic acid complex includes the steps ofcombining an expression cassette with spermidine or spermine with anamphiphilic cationic lipid plus a helper lipid such as cholesterol, anda Fab′ fragment of an antibody attached to a spacer, e.g., polyethyleneglycol, so that the complex has increased shelf life when stored atabout 4° C.

In one particularly preferred embodiment, the method of increasing theshelf life of the lipid:nucleic acid complex includes the steps ofcombining an expression cassette with spermidine or spermine with anamphiphilic cationic lipid, and a Fab′ fragment of an antibody attachedto a polyethylene glycol derivative. In another particularly preferredembodiment, includes the steps of combining an expression cassette withan amphiphilic cationic lipid, and a Fab′ fragment of an antibodyattached to a polyethylene glycol derivative so that the complex hasincreased shelf life when stored at about 4° C.

This invention also provides for a method of transfecting a nucleic acidinto a mammalian cell, the method comprising contacting the cell withany one of the lipid:nucleic acid complexes prepared as described above.In one embodiment, the method uses systemic administration of alipid:nucleic acid complex into a mammal. In a preferred embodiment, themethod of transfecting uses intravenous administration of thelipid:nucleic acid complex into a mammal. In a particularly preferredembodiment, the method comprises contacting a specific cell thatexpresses a ligand that recognizes the Fab′ fragment.

In yet another embodiment, this invention also provides forpharmaceutical composition comprising the lipid:condensed nucleic acidcomplex described above. The pharmaceutical compositions comprise atherapeutically effective dose of the lipid:nucleic acid complex and apharmaceutically acceptable carrier or excipient.

In yet another embodiment, the invention also provides a kit forpreparing a lipid:nucleic acid complex, the kit comprising a containerwith a liposome; a container with a nucleic acid; and a container with ahydrophilic polymer, wherein the liposome and the nucleic acid are mixedto form the lipid:nucleic acid complex and wherein the lipid:nucleicacid complex is contacted with the hydrophilic polymer. In a preferredembodiment, the hydrophilic polymer is derivatized with a targetingmoiety, preferably an Fab′ fragment. In another preferred embodiment,the nucleic acid is condensed.

This invention also provides for a lipid:condensed nucleic acid complexprepared using the method of increasing shelf life using nucleic acidcondensed with an organic polycation, as summarized above.

The invention further provides a method for making lipidicmicroparticles bearing attached proteins. The method employs proteinswhich have been conjugated to linker molecules which will stablyassociate with lipidic microparticles. The invention therefore permitsthe attachment of proteins to the surface, for example, of lipidicmicroparticles which have been preformed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the role of neutral lipid in gene delivery.Three liposome formulations were tested for gene delivery to bothculture cells (SKBR-3, human breast cancer cell) and mice (CD1, female,20-25 g). Samples were: (1) DDAB/Chol (1:1); (2) DDAB/Chol/DOPE(1:0.5:0.5); (3) DDAB/DOPE (1:1); and (4) DDAB alone. FIG. 1Aillustrates cell transfection. SKBR-3 cells were plated at 50,000 cellsper well in twelve-well plates and incubated overnight. Each wellreceived 1 μg of P-CMWIVSLuc+plasmid which was complexed with liposomesat 5 nmole of DDAB. Cells were harvested after 24 hr incubation withcomplexes at 37° C. Values presented are mean from 2 wells. Valuesranged within 10-30% of mean. FIG. 1B illustrates in vivo transfectionin mice. Mice received via tail vein injection 40 μg ofP-CMVIVS-Luc+plasmid, which was complexed with liposomes at 8 nmole DDABper μg DNA ratio. Values presented are mean from 2 mice. Values rangedwithin 20-25% of mean.

FIG. 2 illustrates reporter gene expression in mouse tissue extracts.Mice received (via tail vein injection) 60 μg of P-CMVIVS-Luc+plasmid,which was complexed with DDAB/Chol (1:1) liposomes at 8-nmole DDAB perμg DNA ratio (without spermidine). Values presented are mean from 3mice.

FIG. 3 illustrates the duration of reporter gene expression in mouseluna. Each animal received 40 μg of P-CMVIVS-Luc+plasmid, which wascomplexed with DDAB/Chol (1:1) liposomes at 8 nmole DDAB per μg DNAratio.

FIG. 4 illustrates gene delivery in mouse lung by various stabilizedcomplexes. Each mouse received 60 μg of P-CMVIVS-Luc+, which wascomplexed with DDAB/Chol (1:1) liposomes at 8 nmole DDAB/μg DNA ratio.Values presented are mean from 3 mice. Stippled bars: freshly madecomplexes; filled bars; one month-old samples. Samples are as follows:(1) No stabilizing agent was added; (2) PEG-PE was added at 1% of totallipid to the formed complexes; and (3) Spermidine (0.5 nmole per μg DNA)was added to the plasmid prior to the complex formation.

FIGS. 5A and 5B illustrate in vitro transfection of cell lines withimmunolipid:DNA complexes. The samples are as follows: (1) DDAB/DOPE(1:1), producing cationic liposomes complexed with DNA only; (2)DDAB/DOPE (1:1) with 1% PEG-PE derivatized with maleimide at theultimate position of PEG, producing liposomes with the stericstabilization component added after complexation with the DNA; and (3)DDAB/DOPE (1:1) with 1% PEG-PE derivatized with the Fab′ fragment of ahumanized anti-Her-2 antibody attached to the ultimate position of PEGvia the free thiol group to the maleimide residue.

DEFINITIONS

The following abbreviations are used herein: Chol, cholesterol; PA,phosphatidic acid; PC, phosphatidylcholine; PI, phosphatidylinositol;SM, sphinogmyelin; M-DPE, maleimide derivatized dipalmityolethanolamine;PBS, phosphate buffered saline; LUV, large unilamellar vesicles; MLV,multilamellar vesicles; PE, phosphatidylethanolamine; PEG, polyethyleneglycol; PEG-PE, polyethylene glycol derivatizedphosphatidylethanolamine, DC-chol, 3β [N-(N′, N′-dimethylaminoethane)carbanoyl]-cholesterol; DDAB, Dimethyidioctadecylammonium bromide;DMEPC, Dimyristoylglycero-3-ethyl phosphocholine; DODAP,Dioleoyl-3-dimethylamm onium propane; DOEPC, Dioleoylglycero-3-ethylphosphocholine; DOGS, N,N-Dioctadecylamidoglycyl spermine; DOPE,Dioleoylphosphatidylethanolamine; DOTAP, Dioleoyl-3-trimethylammoniumpropane; DOTMA, N-[2,3-(dioleyloxy) propyl]-N,N,N-trimethyl ammoniumbromide; DSPE, Distearoylphosphatidylethanolamine; PEG-PE,N-[ω-methoxypoly(oxyethylene)-aoxycarbonyl]-DSPE; POEPC,Pahmitoyloleoylglycero-3-ethyl phosphocholine.

The term “amphiphilic cationic lipid” is intended to include anyamphiphilic lipid, including synthetic lipids and lipid analogs, havinghydrophobic and polar head group moieties, a net positive charge, andwhich by itself can form spontaneously into bilayer vesicles or micellesin water, as exemplified by phospholipids. The term also includes anyamphiphilic lipid that is stably incorporated into lipid bilayers incombination with phospholipids with its hydrophobic moiety in contactwith the interior, hydrophobic region of the bilayer membrane, and itspolar head group moiety oriented toward the exterior, polar surface ofthe membrane.

The term “specific binding” refers to that binding which occurs betweensuch paired species as enzyme/substrate, receptor/agonist,antibody/antigen, and lectin/carbohydrate which may be mediated bycovalent or non-covalent interactions or a combination of covalent andnon-covalent interactions. When the interaction of the two speciesproduces a non-covalently bound complex, the binding which occurs istypically electrostatic, hydrogen-bonding, or the result of lipophilicinteractions. Accordingly, “specific binding” occurs between a pairedspecies where there is interaction between the two which produces abound complex having the characteristics of an antibody/antigen orenzyme/substrate interaction. In particular, the specific binding ischaracterized by the binding of one member of a pair to a particularspecies and to no other species within the family of compounds to whichthe corresponding member of the binding member belongs. Thus, forexample, an antibody preferably binds to a single epitope and to noother epitope within the family of proteins.

The terms “ligand” or “targeting moiety”, as used herein, refergenerally to all molecules capable of specifically binding to aparticular target molecule and forming a bound complex as describedabove. Thus the ligand and its corresponding target molecule form aspecific binding pair. Examples include, but are not limited toantibodies, lymphokines, cytokines, receptor proteins such as CD4 andCD8, solubilized receptor proteins such as soluble CD4, hormones, growthfactors, and the like which specifically bind desired target cells, andnucleic acids which bind corresponding nucleic acids through base paircomplementarity. Particularly preferred targeting moieties includeantibodies and antibody fragments (e.g., the Fab′ fragment).

The term “lipid:nucleic acid complex” refers to the product made bymixing amphiphilic cationic lipids or liposomes with a nucleic acid. Theterm “CLDC,” which stands for “cationic lipid:DNA complex” as usedherein is not limited to DNA and is a convenient abbreviation forlipid:nucleic acid complex. The lipid:nucleic acid complex can alsoinclude a helper lipid. The helper lipid is often a neutral lipid suchas DOPE or cholesterol with cholesterol being most preferred. Thelipid:nucleic acid complex may also contain other compounds such as apolycation that are in contact with the nucleic acid of the complex,producing condensed nucleic acid, and hydrophilic polymers such as PEGand derivatized PEG.

The terms “immunoliposome” and “immunolipid:nucleic acid complex” referto a liposome or lipid:nucleic acid complex bearing an antibody orantibody fragment that acts as a targeting moiety enabling thelipid:nucleic acid complex to specifically bind to a particular “target”molecule that may exist in solution or may be bound to the surface of acell. Where the target molecule is one that is typically found inrelative excess (e.g., ≧10-fold) and in association with a particularcell type or alternatively in a multiplicity of cell types allexpressing a particular physiological condition the target molecule issaid to be a “characteristic marker” of that cell type or thatphysiological condition. Thus, for example, a cancer may becharacterized by the overexpression of a particular marker such as theHER2 (c-erbB-21neu) proto-oncogene in the case of breast cancer.

A “hydrophilic polymer” as used herein refers to highly hydratedflexible neutral polymers attached to lipid molecules. Examples include,but are not limited to polyethylene glycol (PEG), polyethylene glycolderivatized with phosphatidyl ethanolamine (PEG-PE), polyethylene glycolderivatized with tween, polyethylene glycol derivatized withdistearoylphosphatidylethanolamine (PEG-DSPE), ganglioside GMI andsynthetic polymers. Such polymers typically have a molecular weight inthe range of 1000-10,000. Preferably, the molecular weight for PEG isapproximately 2000.

“Transfection” refers to contacting a living cell with a nucleic acid,for example, as part of a lipid:nucleic acid complex.

“Transfection activity” refers to the efficiency of introducing anucleic acid into a living cell. Transfection efficiency may be measuredby determining the amount of expression of a reporter gene that has beentransfected into the cell as part of a lipid:nucleic acid complex, forexample, by fluorescent or functional assays.

The terms “condensed nucleic acid” and “partially condensed nucleicacid” are used to refer to a nucleic acid that has been contacted withan organic cation for example, polyamines, including spermine andspermidine, polyammonium molecules such as Polybrene (hexadimethrinebromide), basic polyamino acids, and basic proteins. Condensed nucleicacids typically occupy a significantly smaller volume than non-condensednucleic acids. It is recognized, however, that the degree ofcondensation may vary with local environment (e.g., lipid as opposed toaqueous environment).

The term “shelf life” when used to refer to the lipid:nucleic acidsdisclosed herein refers to the period of time the lipid:nucleic acidcomplex can be stored (under defined conditions e.g., at 4° C.) beforelosing its biological activity. The biological activity assayed fordetermination of shelf life in the present invention is the ability ofthe lipid:nucleic acid complex to transfect mammalian cells in vivoafter intravenous administration. The “shelf life” of a lipid:nucleicacid complex is conveniently determined by assaying by gene expressionfrom reporter nucleic acids in the lipid:nucleic acid complex asdescribed herein.

An “expression cassette” refers to a promoter operably linked to a DNAmolecule, containing all the elements required for expression of thatDNA molecule in a living cell. The expression cassette may containadditional elements such as enhancers, replication origins and the like,forming an expression vector. “Organic polycation” or “polycation” referto a an organic polymeric structure where more than one unit of thepolymer bears a negative charge and the net charge of the polymer ispositive. Examples of such an organic cation are polyamines, includingspermine and spermidine, polyammonium molecules such as Polybrene(hexadimethrine bromide), basic polyamino acids, or basic proteins.

A “pharmaceutically acceptable carrier” is a material that is notbiologically or otherwise undesirable, i.e., the material can beadminister to an individual along with the lipid:nucleic acid complexwithout causing unacceptable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

The term “nucleic acid” refers to a polymer or oligomer composed ofnucleotide units (ribonucleotides, deoxyribonucleotides or relatedstructural variants or synthetic analogs thereof) linked viaphosphodiester bonds (or related structural variants or syntheticanalogs thereof). Thus, the term refers to a nucleotide polymer in whichthe nucleotides and the linkages between them are naturally occurring(DNA or RNA), as well as various analogs, for example and withoutlimitation, peptide-nucleic acids (PNAs), phosphoramidates,phosphorothioates, methyl phosphonates, 2-O-methyl ribonucleic acids,and the like.

The term “mole percent” when referring to the percentage of hydrophilicpolymer in a liposome is expressed relative to the cationic lipid in theliposome unless otherwise stated. Thus, for example, in a liposomecomprising a ratio of DDAB to cholesterol (Chol) of 100:100, a 4 molepercent of hydrophilic polymer (e.g., PEG) would represent a ratio ofDDAB:Chol:PEG of about 100:100:4.

The term “identical” refers to a composition that is formed using thesame compounds as another composition, where the compositions do notdiffer in a statistically significant manner.

The term “systemic administration” refers to a method of administering acompound or composition to a mammal so that the compound or compositionis delivered to many sites in the body via the circulatory system.

As used herein, “linker molecule” means a molecule comprising (a) ahydrophobic domain, (b) a hydrophilic polymer chain terminally attachedto the hydrophobic domain, and (c) a chemical group reactive to one ormore functional groups on a protein molecule and attached to the saidpolymer chain at, or near to, the terminus contralateral to thehydrophobic domain.

The term “hydrophobic domain” of, for example, a linker molecule, meansa fatty acid, fatty alcohol, sterol, or other hydrophobic moleculecapable of distribution into a lipid phase from an aqueous medium. Forexample, a hydrophobic domain may be a diacylglycerol, a phospholipid, asterol, such as cholesterol, or a diacylamide derivative, such asN,N-distearoyl-glycineamide.

The term “lipid moiety” with reference to a protein molecule means anarray composed of one or more hydrophobic domains directly covalentlybound to the protein molecule.

The terms “protein” and “peptide” are generally differentiated in theart by molecular weight, with polypeptides below 6,000 Daltons beingconsidered peptides and those at or above 6,000 Daltons being consideredproteins. See, e.g., McMurray, Organic Chemistry (Brooks/Cole PublishingCo., Belmont, Calif.)(1988), at p. 971. The use of these terms hereinfollows this distinction.

DETAILED DESCRIPTION

This invention provides methods of increasing the shelf life of cationiclipid:nucleic acid complexes, and in vivo and/or in vitro transfectionefficiency of these complexes. Such complexes have attractedconsiderable interest as a means of delivering nucleic acids expressingvarious therapeutic polypeptides as a means of delivering therapeutic(e.g., antisense) nucleic acids themselves. Unfortunately, it has beendifficult to maintain and store homogeneous lipid:nucleic acid complexessuitable for in vivo administration. The complexes tend to aggregaterapidly or decompose within a relatively short time. This instabilityhas required use of these complexes within a short period of time afterpreparation, often as little as 30 minutes up to a few hours. Thus, forexample, in recent clinical trials using cationic lipids as a carrierfor DNA delivery, the DNA and lipid components were mixed at the bedsideand used immediately (Gao et al., Gene Therapy 2: 710-722 (1995)).

This lipid:nucleic acid complex instability provides a significanthindrance to the widespread acceptance of cationic lipid:nucleic acidcomplexes as therapeutics. The necessity of preparation of the complexshortly before use requires that a pharmaceutical facility be inrelatively close proximity to the area of use. Alternatively,combination of the lipid and nucleic acid at the bedside imposes asubstantial labor burden, introduces quality control problems ininsuring adequate complexation, and creates a source of potential error.

The present invention solves these problems by providing methods ofsignificantly increasing the shelf (storage) life of lipid:nucleic acidcomplexes. The methods generally involve: (1) condensing the nucleicacid before incorporation into the lipid:nucleic acid complex; (2)combining a lipid:nucleic acid complex with a hydrophilic polymer (e.g.,PEG); and (3) both condensing the nucleic acid prior to complexformation and combining the complex with a hydrophilic polymer.

While condensation of nucleic acids may lead to stability of the nucleicacid in isolation (e.g., in an aqueous buffer), it was a surprisingdiscovery of this invention that the use of a condensing agent (e.g., anorganic polycation) provides a lipid:nucleic acid complex that remainscapable of transfecting a cell in vivo even after a period of prolongedstorage (e.g., cold storage at a temperature of about 22° C. or below,more preferably ranging from about 0° C. to about 22° C., and mostpreferably at about 4° C.).

It was also a surprising discovery that lipid:nucleic acid complexescombined with a hydrophilic polymer attached to an amphipathic lipid(e.g., PEG-PE) also show an increased shelf life. Without being bound bya particular theory, it is believed that when the cationic lipid:DNAcomplex (“CLDC”) is contacted with the hydrophilic polymer, thehydrophilic polymer locates and is incorporated into hydrophobic pocketsin the complex via its hydrophobic side chains, while leaving thehydrophilic part at the exterior surface, thereby stabilizing the entirecomplex.

In view of these discoveries, this invention provides methods ofincreasing the shelf life of cationic lipid:nucleic acid complexes. Themethods generally involve either condensing the nucleic acid using apolycation and/or contacting, e.g., coating, the lipid:nucleic acidcomplex with a hydrophilic polymer. This invention also include thelipid:nucleic acid complexes thus prepared.

This invention further provides methods for forming lipidicmicroparticles with attached proteins suitable, for example, fortargeting the microparticles to selected cells or tissues. The methodsprovide a number of advantages over prior art methods:

(1) due to the quantitative nature of insertion, the number of proteinsper particle is highly reproducible and can be precisely defined;

(2) more than one kind of protein can be attached to the surface of thesame particle, in a precise proportion;

(3) if the protein-linker conjugate is purified before insertion into aparticle surface, the particle will not bear unconjugated linkers;

(4) the particle may contain, in its composition, molecules reactivewith the linker active group. For example, the particle may containthiols, even if the active group is maleimide;

(5) if the particle is a vesicle, the linker/protein molecules will onlybe present on the outer surface; and,

(6) the method increases the utility of premade, known particles, suchas commercially made pharmaceutical liposomes, by permitting theaddition of surface-attached conjugates bearing proteins of interest.

I. Cationic lipid:nucleic acid complexes

As explained above, this invention provides methods of increasing thestorage life (shelf life) of lipid:nucleic acid complexes. In apreferred embodiment the complexes are formed by combination of anucleic acid with a liposome. It is recognized, however, that the lipidsneed not be provided as a liposome. It is also recognized that aftercomplexation, the lipid:nucleic acid complex may no longer exist as atrue vesicle and therefore is not generally regarded as a liposome. Thepreparation of lipid:nucleic acid complexes is well known to one ofskill in the art (see, e.g., reviewed in Crystal, Science 270: 404-410(1995); Blaese et al., Cancer Gene Ther. 2: 291-297 10 (1995); Behr etal., Bioconjugate Chem. 5: 382-389 (1994); Remy et al., BioconjugateChem. 5: 647-654 (1994); and Gao et al., Gene Therapy 2: 710-722(1995)). The various components and construction of the stabilizedlipid:nucleic acid complexes of the invention are described in detailbelow.

A. Amphiphilic cationic lipids

As indicated above, the methods of this invention involve complexing acationic lipid with a nucleic acid. The term “cationic lipid” refers toany of a number of lipid species which carry a net positive charge atphysiological pH. Such lipids include, but are not limited to, DODAC,DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. Additionally, a number ofcommercial preparations of cationic lipids are available which can beused in the present invention. These include, for example, LIPOFECTIN®(commercially available cationic liposomes comprising DOTMA and DOPE,from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE® (commerciallyavailable cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL);and TRANSFECTAM® (commercially available cationic lipids comprising DOGSin ethanol from Promega Corp., Madison, Wis., USA).

The cationic lipid can be used alone, or in combination with a “helper”lipid. Preferred helper lipids are non-ionic or uncharged atphysiological pH. Particularly preferred non-ionic lipids include, butare not limited to cholesterol and DOPE, with cholesterol being mostpreferred.

The molar ratio of cationic lipid to helper can range from 2:1 to about1:2, more preferably from about 1.5:1 to about 1:1.5 and most preferablyis about 1:1. Additional cationic and nonionic lipids suitable for usein the lipid:nucleic acid complexes of the present invention are wellknown to persons of skill in the art and are cited in a variety of wellknown sources, e.g., McCutcheon's Detergents and Emulsifiers andMcCutcheon's Functional Materials, Allured Publishing Co., Ridgewood,N.J. Preferred lipids include DDAB:cholesterol or DOTAP:cholesterol at amolar ratio of 1:1.

B. Nucleic acid

The lipid:nucleic acid complexes contain a nucleic acid, typically anexpression cassette that is constructed using recombinant techniques. Arecombinant nucleic acid is prepared by first isolating the nucleic acidof interest. The isolated nucleic acid is then ligated into a cassetteor vector suitable for expression of the gene. Methods for preparing arecombinant nucleic acid are known by those skilled in the art (seeSambrook et al., Molecular Cloning: A Laboratory Manual (2d ed. 1989)).

The gene of interest, for example, a gene encoding a therapeuticpolypeptide, or a reporter gene, can be inserted into an “expressionvector,” “cloning vector,” or “vector,” terms which usually refer toplasmids or other nucleic acid molecules that are able to replicate in achosen host cell and express the gene of interest. Expression vectorscan replicate autonomously, or they can replicate by being inserted intothe genome of the host cell. Often, it is desirable for a vector to beusable in more than one host cell, e.g., in E. coli for cloning andconstruction, and in a mammalian cell for expression. Additionalelements of the vector can include, for example, selectable markers andenhancers. Selectable markers, e.g., tetracycline resistance orhygromycin resistance, permit detection and/or selection of those cellstransformed with the desired DNA sequences (see, e.g., U.S. Pat. No.4,704,362). The particular vector used to transport the geneticinformation into the cell is also not particularly critical. Any of theconventional vectors used for expression of recombinant proteins inprokaryotic or eukaryotic cells can be used.

The expression vectors typically have a transcription unit or expressioncassette that contains all the elements required for the expression ofthe nucleic acid in the host cells. A typical expression cassettecontains a promoter operably linked to the DNA sequence encoding aprotein. The promoter is preferably positioned about the same distancefrom the heterologous transcription start site as it is from thetranscription start site in its natural setting. As is known in the art,however, some variation in this distance can be accommodated withoutloss of promoter function.

In the expression cassette, the nucleic acid sequence of interest can belinked to a sequence encoding a cleavable signal peptide sequence topromote secretion of the encoded protein by the transformed cell. Theexpression cassette should also contain a transcription terminationregion downstream of the structural gene to provide for efficienttermination. The termination region can be obtained from the same geneas the promoter sequence or can be obtained from a different gene.

For more efficient translation in mammalian cells of the mRNA encoded bythe structural gene, polyadenylation sequences are also commonly addedto the expression cassette. Termination and polyadenylation signals thatare suitable for the present invention include those derived from SV40,or a partial genomic copy of a gene already resident on the expressionvector.

In addition to the expression cassette, many expression vectorsoptimally include enhancer elements that can stimulate transcription upto 1,000 fold from linked homologous or heterologous promoters. Manyenhancer elements derived from viruses have a broad host range and areactive in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus, the longterminal repeat from various retroviruses such as murine leukemia virus,murine or Rous sarcoma virus, and HIV (see Enhancers and EukaryoticExpression (1983)).

In addition to the recombinant nucleic acids discussed above, syntheticnucleic acids or oligonucleotides can also be used in the invention. Asa general point regarding the nucleic acids used in the invention, thoseof skill in the art recognize that the nucleic acids used in theinvention include both DNA and RNA molecules, as well as synthetic,non-naturally occurring analogs of the same, and heteropolymers, ofdeoxyribonucleotides, ribonucleotides, and/or analogues of either. Theparticular composition of a nucleic acid or nucleic acid analogue willdepend upon the purpose for which the material will be used and theenvironment in which the material will be placed. Modified or synthetic,non-naturally occurring nucleotides have been designed to serve avariety of purposes and to remain stable in a variety of environments,such as those in which nucleases are present, as is well known in theart. Modified or synthetic non-naturally occurring nucleotides, ascompared to naturally occurring ribo- or deoxyribonucleotides may differwith respect to the carbohydrate (sugar), phosphate bond, or baseportions of the nucleotide, or may even contain a non-nucleotide base(or no base at all) in some cases (see, e.g., Arnold et al., PCT patentpublication no. WO 89/02439). For example, the modified or non-naturallyoccurring nucleic acids of the invention can include biotinylatednucleic acids, O-methylated nucleic acids, methylphosphonate backbonenucleic acids, phosphorothioate backbone nucleic acids, or polyamidenucleic acids.

Oligonucleotides, such as antisense RNA described below, preferably aresynthesized on an Applied BioSystems or other commercially availableoligonucleotide synthesizer according to specifications provided by themanufacturer. Oligonucleotides may be prepared using any suitablemethod, such as the phosphotriester and phosphodiester methods, orautomated embodiments thereof. In one such automated embodiment,diethylphosphoramidites are used as starting materials and may besynthesized as described by Beaucage et al., Tetrahedron Letters 22:1859 (1981), and U.S. Pat. No. 4,458,066.

C. Condensed nucleic acid

Small polycationic molecules are known to condense nucleic acids viaelectrostatic charge-charge interactions (Plum et al., Biopolymers 30:631-643 (1990)). The pretreatment of nucleic acid with polyamines cantherefore reduce the number of charge sites for complexing with cationicliposomes. However, condensing nucleic acid prior to lipid complexformation produced the surprising result of increased shelf life forlipid:nucleic acid complexes, as measured by transfection efficiency.The lipid:nucleic acid complexes formed with such pretreatment werestable at a lower ratio of lipid to DNA without aggregation. Organicpolycations such as polyamines, polyammonium molecules, and basicpolyamino acids, and their derivatives are used to condense the nucleicacid prior to lipid complex formation. A preferred embodiment usespolyamines such as spermidine and spermine to condense the nucleic acid(see, e.g., Example 1).

D. Hydrophilic polymer

It has been established recently that PEG-PE incorporation in liposomesproduces steric stabilization resulting in longer circulation times inblood (Allen et al., Biochim. Biophys. Acta 1066: 29-36 (1991);Papahadjopoulos et al., Proc. Natl. Acad. Sci. USA 88: 11460-11464(1991)). In the present invention, inserting PEG-PE (e.g., 1% of totallipid) into the freshly formed lipid:nucleic acid complexes prevents thecomplexes from aggregating during storage. It was a surprisingdiscovery, however, that the incorporation of PEG-PE did not inhibittransfection activity in vivo and also that the in vitro transfectionactivity, which was inhibited, was regained by the incorporation of Fab′fragment conjugated at the end of the PEG-PE. The presence ofhydrophilic polymers in the lipid:nucleic acid complex providesincreased shelf life, as measured by transfection efficiency afterstorage. Thus, it is desirable to add a hydrophilic polymer such aspolyethylene glycol (PEG)-modified lipids or ganglioside G_(M1) to theliposomes. PEG may also be derivatized with other amphipathic moleculessuch as fatty acids, sphingolipids, glycolipids, and cholesterol.Addition of such components prevents liposome aggregation duringcoupling of the targeting moiety to the liposome. These components alsoprovide a means for increasing circulation lifetime of the lipid:nucleicacid complexes.

A number of different methods may be used for the preparation of PEG forincorporation into liposomes. In one preferred embodiment, PEG isincorporated as PEG derivatized phosphatidylethanolamine (PEG-PE) or PEGderivatized distearoyl phosphatidylethanolamine (PEG-DSPE). Methods ofpreparing PEG-PE are well known and typically involve using an activatedmethoxy PEG (with only one reactive end) and PE. Thus PEG-succinimidylsuccinate may be reacted in a basic organic solvent (Klibanov et al.,FEBS Lett. 268: 235-237 (1990)). A particularly preferred method ofPEG-PE preparation is based on reaction of the PEG withcarbonyldiimidazole followed by addition of PE (see Woodle et al., Proc.Intern. Symp. Control. Rel. Bioact. Mater. 17: 77-78 (1990);Papahadjopoulos et al., Proc. Natl. Acad. Sci. USA 88: 11460-11464(1991); Allen et al., Biochim. Biophys. Acta. 1066: 29-36 (1991); Woodleet al., Biochim. Biophys. Acta 1105: 193-200 (1992); and Woodle et al.,Period. Biol. 93: 349-352 (1991)). Similarly, cyanuric chlorideactivated PEG in a basic organic solvent is described by Blume et al.,Biochim. Biophys. Acta. 1029: 91-97 (1990) and U.S. Pat. No. 5,213,804.A completely different approach is based on coupling the PEG withpreformed liposomes utilizing tresyl chloride activated PEG which isthen added to liposomes containing PE at high pH (Senior et al.,Biochim. Biophys. Acta. 1062: 77-82 (1991)). Derivatized PEG is alsocommercially available. Thus, for example, PEG-PE is available fromAvanti Polar lipids (Alabaster, Ala.). One of skill in the art willrecognize that many other linkages are available, e.g., PEG linkeddetergents such as tweens and insertion of PEG derivatized lipid intoformed lipid:nucleic acid complexes.

E. Fab′ antibody fragment

In a preferred embodiment, the lipid:nucleic acid complexes of thepresent invention are conjugated to the Fab′ fragment of an antibody,which acts as a targeting moiety enabling the lipid:nucleic acid complexto specifically bind a target cell bearing the target molecule (e.g.,characteristic marker) to which the Fab′ antibody fragment is directed.Smaller peptides from the hypervariable region or from another peptideinteracting with a specific cell surface ligand may also be conjugatedto the complexes. In general terms, the Fab′ fragment of an antibodyrepresents a monomer comprising the variable regions and the C_(H)1region of one arm of an antibody. One such preferred embodiment isdescribed in Example 2.

An “antibody” refers to a protein consisting of one or more polypeptidessubstantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

The basic immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies may exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.In particular, pepsin digests an antibody below the disulfide linkagesin the hinge region to produce F(ab)′₂, a dimer of Fab′ which itself isa light chain joined to V_(H)-C_(H)1 by a disulfide bond. The F(ab)′₂may be reduced under mild conditions to break the disulfide linkage inthe hinge region thereby converting the F(ab)′₂ dimer into an Fab′monomer. The Fab′ monomer is essentially an Fab with part of the hingeregion (see Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y.(1993) for more antibody fragment terminology). While the Fab′ fragmentis defined in terms of the digestion of an intact antibody, one of skillwill appreciate that such Fab′ fragments may be synthesized de novoeither chemically or by utilizing recombinant DNA methodology.

The Fab′ fragments used in the present invention may be derived fromantibodies of animal (especially mouse or rat) or human origin or may bechimeric (Morrison et al., Proc Natl. Acad. Sci. USA 81: 6851-6855(1984)) or humanized (Jones et al., Nature 321: 522-525 (1986), andpublished UK patent application No. 8707252).

The Fab′ fragment is selected to specifically bind to a molecule ormarker characteristic of the surface of the cells to which it is desiredto deliver the contents of the cationic lipid:nucleic acid complex. Amolecule is characteristic of cell, tissue, or physiological state whenthat molecule is typically found in association with that cell type oralternatively in a multiplicity of cell types all expressing aparticular physiological condition (e.g., transformation). A specificcharacteristic marker is preferably found on the surfaces of cells of aparticular tissue or cell type or on the surfaces of tissues or cellsexpressing a particular physiological condition and on no other tissueor cell type in the organism. One of skill will recognize however, thatsuch a level of specificity of the marker is often not required. Forexample a characteristic cell surface marker will show sufficient tissuespecificity if the only non-target tissues are not accessible to thelipid:nucleic acid complex. Alternatively, effective specificity may beachieved by overexpression of the marker in the target tissue ascompared to other tissues. This will result in preferential uptake bythe target tissue leading to effective tissue specificity. Thus forexample, many cancers are characterized by the overexpression of cellsurface markers such as the HER2 (c-erbB-2, neu) proto-oncogene encodedreceptor in the case of breast cancer.

One of skill will recognize that there are numerous cell surface markersthat provide good characteristic markers depending on the particulartissue it is desired to target. These cell surface markers include, butare not limited to carbohydrates, proteins, glycoproteins, MHCcomplexes, interleukins, and receptor proteins such as HER, CD4 and CD8receptor proteins as well as other growth factor and hormone receptorproteins.

Growth factor receptors are particularly preferred characteristic cellsurface markers. Growth factor receptors are cell surface receptors thatspecifically bind growth factors and thereby mediate a cellular responsecharacteristic of the particular growth factor. The term “growthfactor”, as used herein, refers to a protein or polypeptide ligand thatactivates or stimulates cell division or differentiation or stimulatesbiological response like motility or secretion of proteins. Growthfactors are well known to those of skill in the art and include, but arenot limited to, platelet-derived growth factor (PDGF), epidermal growthfactor (EGF), insulin-like growth factor (IGF), transforming growthfactor β (TGF-β), fibroblast growth factors (FGF), interleukin 2 (IL2),nerve growth factor (NGF), interleukin 3 (IL3), interleukin 4 (IL4),interleukin 1 (IL1), interleukin 6 (IL6), interleukin 7 (IL7),granulocyte/macrophage colony-stimulating factor (GM-CSF), granulocytecolony-stimulating factor (G-CSF), macrophage colony-stimulating factor(M-CSF), erythropoietin, interleukin 13 receptor (IL13R), and the like.One of skill in the art recognizes that the term growth factor as usedherein generally includes cytokines and colony stimulating factors.

Particularly preferred markers are found in the HER family of growthfactor receptors. More specifically HER1, HER2, HER3 and HER4 are morepreferred with HER2 most preferred. The HER receptors comprise proteintyrosine kinases that themselves provide highly specific antibodytargets. Thus, in one embodiment, the P185 tyrosine kinase of HER2provides a most preferred target for the Fab′ fragment of the utilizedin the immunolipid:nucleic acid complexes of the present invention.

It will be appreciated that the characteristic marker need not be anaturally occurring marker, but rather may be introduced to theparticular target cell. This may be accomplished by directly tagging acell or tissue with a particular marker (e.g., by directly injecting theparticular target tissue with a marker, or alternatively, byadministering to the entire organism a marker that is selectivelyincorporated by the target tissue. In one embodiment, the marker may bea gene product that is encoded by a nucleic acid in an expressioncassette. The marker gene may be under the control of a promoter that isactive only in the particular target cells. Thus introduction of avector containing the expression cassette will result in expression ofthe marker in only the particular target cells. One of skill in the artwill recognize that there are numerous approaches utilizing recombinantDNA methodology to introduce characteristic markers into target cells.

In one preferred embodiment, the targeting moiety will specifically bindproducts or components of a growth factor receptor, in particularproducts of the HER2 (c-erbB-2, neu) proto-oncogene. It is particularlypreferred that the targeting moiety bind the growth factorreceptor-tyrosine kinase encoded by HER2, protein p185^(HER2), which iscommonly overexpressed in breast cancers (Slamon et al., Science 235:177-182 (1987). Other suitable targets for the targeting moiety include,but are not limited to EGFR (HER1), HER3, and HER4, combinations ofthese receptors, and other markers associated with cancers. Otherantibodies of interest include, but are not limited to BR96 (Friedman etal., Cancer Res. 53: 334-339 (1993), e23 to erbB2 (Batra et al, Proc.Natl. Acad. Sci. USA 89: 5867-5871 (1992)), PR1 in prostate cancer(Brinklann et al., Proc. Natl. Acad. Sci. USA. 90: 547-551 (1993)), andK1 in ovarian cancer (Chang et al. Int. J. Cancer 50: 373-381 (1992).

Immunolipid:nucleic acid complexes of the present invention may beprepared by incorporating the Fab′ fragment into the liposomes or lipidsby a variety of techniques well known to those of skill in the art. TheFab′ is added to the lipid:nucleic acid complex either before or aftercomplex formation. For example, a biotin conjugated Fab′ may be bound toa liposome containing a streptavidin. Alternatively, the biotinylatedFab′ may be conjugated to a biotin derivatized liposome by an avidin orstreptavidin linker. Thus, for example, a biotinylated monoclonalantibody was biotinylated and attached to liposomes containingbiotinylated phosphatidylethanolamine by means of an avidin linker (see,e.g., Ahmad et al., Cancer Res. 52: 4817-4820 (1992)). Typically about30 to 125 and more typically about 50 to 100 Fab′ fragments perlipid:nucleic acid complex are used.

In a preferred embodiment, the targeting moiety may be directlyconjugated to the liposome. Such means of direct conjugation are wellknown to those of skill in the art (see, e.g., Gregoriadis, LiposomeTechnology (1984) and Lasic, Liposomes: from Physics to Applications(1993)). Particularly preferred is conjugation through a thioetherlinkage. This may be accomplished by reacting the antibody with amaleimide derivatized lipid such as maleimide derivatizedphosphatidylethanolamine (M-PE) or dipalmitoylethanolamine (M-DEP). Thisapproach is described in detail by Martin et al. J. Biol. Chem. 257:286-288 (1982).

II. Preparation of liposomes

A variety of methods are available for preparing liposomes as describedin, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980); U.S.Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,946,787; PCT Publication No. WO91/17424; Szoka & Papahadjopoulos, Proc. Natl. Acad. Sci. USA 75:4194-4198 (1978); Deamer & Bangham, Biochim. Biophys. Acta 443: 629-634(1976); Fraley et al., Proc. Natl. Acad. Sci. USA 76: 3348-3352 (1979);Hope et al., Biochim. Biophys. Acta 812: 55-65 (1985); Mayer et al.,Biochim. Biophys. Acta 858: 161-168 (1986); Williams et al., Proc. Natl.Acad. Sci. USA 85: 242-246 (1988), Liposomes, ch. 1 (Ostro, ed., 1983);and Hope et al., Chem. Phys. Lip. 40: 89 (1986). Suitable methodsinclude, e.g., sonication, extrusion, high pressure/homogenization,microfluidization, detergent dialysis, calcium-induced fusion of smallliposome vesicles, and ether-infusion methods, all well known in theart. One method produces multilamellar vesicles of heterogeneous sizes.In this method, the vesicle-forming lipids are dissolved in a suitableorganic solvent or solvent system and dried under vacuum or an inert gasto form a thin lipid film. If desired, the film may be redissolved in asuitable solvent, such as tertiary butanol, and then lyophilized to forma more homogeneous lipid mixture which is in a more easily hydratedpowder-like form. This film is covered with an aqueous buffered solutionand allowed to hydrate, typically over a 15-60 minute period withagitation. The size distribution of the resulting multilamellar vesiclescan be shifted toward smaller sizes by hydrating the lipids under morevigorous agitation conditions or by adding solubilizing detergents suchas deoxycholate.

In a preferred embodiment, mostly unilammellar liposomes are produced bythe reverse phase evaporation method of Szoka & Papahadjopoulos, Proc.Natl. Acad. Sci. USA, 75: 4194-4198 (1978).

Unilamellar vesicles are generally prepared by sonication or extrusion.Sonication is generally performed with a bath-type sonifier, such as aBranson tip sonifier at a controlled temperature as determined by themelting point of the lipid. Extrusion may be carried out by biomembraneextruders, such as the Lipex Biomembrane Extruder. Defined pore size inthe extrusion filters may generate unilamellar liposomal vesicles ofspecific sizes. The liposomes may also be formed by extrusion through anasymmetric ceramic filter, such as a Ceraflow Microfilter, commerciallyavailable from the Norton Company, Worcester Mass.

Following liposome preparation, the liposomes that have not been sizedduring formation may be sized by extrusion to achieve a desired sizerange and relatively narrow distribution of liposome sizes. A size rangeof about 0.2-0.4 microns allows the liposome suspension to be sterilizedby filtration through a conventional filter, typically a 0.22 micronfilter. The filter sterilization method can be carried out on a highthrough-put basis if the liposomes have been sized down to about 0.2-0.4microns.

Several techniques are available for sizing liposomes to a desired size.One sizing method is described in U.S. Pat. Nos. 4,529,561 or 4,737,323.Sonicating a liposome suspension either by bath or probe sonicationproduces a progressive size reduction down to small unilamellar vesiclesless than about 0.05 microns in size. Homogenization is another methodwhich relies on shearing energy to fragment large liposomes into smallerones. In a typical homogenization procedure, multilamellar vesicles arerecirculated through a standard emulsion homogenizer until selectedliposome sizes, typically between about 0.1 and 0.5 microns, areobserved. The size of the liposomal vesicles may be determined byquasi-electric light scattering (QELS) as described in Bloomfield, Ann.Rev. Biophys. Bioeng., 10: 421-450 (1981). Average liposome diameter maybe reduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Extrusion of liposome through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is also an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in liposome size. For use in the presentinvention, liposomes having a size of about 0.05 microns to about 0.5microns. More preferred are liposomes having a size of about 0.05 to 0.2microns.

III. Formation of lipid:nucleic acid complexes

It was a discovery of this invention that stabilized lipid:nucleic acidcomplexes (e.g., having condensed nucleic acid and/hydrophilic polymer)tended not to form visible large aggregates and had increasedtransfection efficiency and shelf life. Nucleic acid/liposome ratios forpreparing lipid:nucleic acid complexes that do not form visible largeaggregates can be determined by one of skill in the art. Typically, theratio is determined by mixing fixed amounts of a nucleic acid, e.g., aplasmid, to various amounts of liposomes (see Example 1). In general,lipid:nucleic acid complexes are formed by pipetting the nucleic acid(e.g., plasmid DNA) into a liposome suspension of equal volume andmixing rapidly. Routinely, liposomes containing 5-15 nmole of a lipidsuch as DDAB or DOPE (as described above) form a complex with 1 μgplasmid, without forming visible large aggregates. Inspection forvisible large aggregates is typically performed without the aid of amicroscope. The endpoint of the titration of the amounts of lipid andnucleic acid is also achieved by assaying for increased transfectionefficiency, either in vitro or in vivo, as compared to a non-stabilizedcontrol (as described below).

To keep the lipid:nucleic acid complexes from forming large aggregatesand losing transfecting activity with time, two approaches are taken:(1) incorporating a small amount of a hydrophilic polymer such as PEG-PE(approx. 1% mole ratio) into lipid:nucleic acid complexes within a fewminutes after their preparation; and/or (2) condensing the nucleic acidwith a polycation such as a polyamine (e.g., approximately 0.05 to 5.0nmole of spermidine per μg DNA) prior to mixing with the liposomes. Theoptimal amount of the polyamines and hydrophilic polymer can bedetermined by one of skill in the art by titrating the polyamine orhydrophilic polymer with the nucleic acid so that the formed complexesdo not form large, e.g., visible, aggregates. The size of theselipid:nucleic acid complexes can be estimated by dynamic lightscattering to be in the range of 410±150 nm. The endpoint of thetitration is also achieved by assaying for increased transfectionefficiency either in vitro or in vivo, as compared to a non-stabilizedcontrol (as described below).

IV. Transfection and gene therapy with lipid:nucleic acid complexes

The present invention provides lipid:nucleic acid complexes that haveincreased shelf life, for transfection of mammalian cells in vitro, invivo, and ex vivo, and methods of production and transfection of suchcomplexes. In particular, this invention relies in part on theunexpected discovery that a lipid:nucleic acid complex comprisingnucleic acid that has been condensed by contact with an organicpolycation demonstrates an increased shelf life. In addition, thisinvention relies on the unexpected discovery that a lipid:nucleic acidcomplex, which is mixed with a hydrophilic polymer after lipid:nucleicacid complex formation, exhibits high transfection activity andincreased shelf life, as measured by transfection activity afterstorage. Such lipid:nucleic acid complexes having increased shelf lifeare useful, e.g., for in vitro and ex vivo transfection of cells, andfor delivery of nucleic acids into cells for mammalian gene therapy invivo and following intravenous administration.

Using lipid:nucleic acid complexes to deliver nucleic acids intodifferent mammalian cell types results in a safe method of transfer, andhigh efficiency of gene transfer. Transfection of cells in vivo withlipid:nucleic acid complexes is known to those skilled in the art andcan be performed using standard techniques, as discussed in Example 1(see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2ded. 1989); Ausubel et al., Current Protocols in Molecular Biology(1995)).

Any heterologous nucleic acid that is suitable for introduction into ahost cell can be used in the present invention by one skilled in theart. Genes useful for gene therapy can be introduced into mammals usingthe methods and vectors of this invention. Genes encoding bloodproteins, enzymes, hormones, ribozymes, antisense RNA, viral inhibitors,and ion channel proteins are examples of heterologous nucleic acidsuseful in gene therapy. A functional heterologous gene can be used toreplace a mutated gene in a mammal using gene therapy. For example, thegene encoding β-globin can be used to treat β-thalassemia; and the geneencoding CFTR can be used to treat cystic fibrosis. Genes encodingselectable markers, such as those that confer antibiotic resistance, canbe used to detect and isolate cells transfected with the lipid:nucleicacid complex. Reporter genes such as luciferase, ,-galactosidase,chloramphenicol acetyl transferase (CAT), human growth hormone (hGH),and the green fluorescent protein (GFP) are preferred examples of genesthat can be used in assays to determine transfection efficiency. In oneembodiment of the invention, luciferase can be used as a reporter geneto determine transfection efficiency.

Transfection efficiency of a reporter gene can be determined with anassay that is appropriate for the reporter gene in use. Such assays areknown to those skilled in the art. For example, the HGH reporter assayis immunologically based and employs commercially availableradioimmunoassay kits. In a preferred embodiment of the invention, theluciferase assay is used to detect transfection and expression of theluciferase reporter gene. The luciferase assay is preferred because itis highly sensitive and does not use radioactivity. A luminometer can beused to measure the luciferase enzyme activity, as described in Example1.

Gene therapy provides methods for combating chronic infectious diseasessuch as HIV infection, as well as non-infectious diseases such as cancerand birth defects (see generally Anderson, Science 256: 808-813 (1992);Yu et al., Gene Ther. 1: 13-26 (1994)). Gene therapy can be used totransduce cells with either an ex vivo or an in vivo procedure. Ex vivomethods for gene therapy involve transducing the cell outside of themammal with a lipid:nucleic acid complex of this invention, andintroducing the cell back into the organism. The cells can behematopoietic stem cells isolated from bone marrow or other cells thatcan be transfected by lipid:nucleic acid complexes.

In humans, hematopoietic stem cells can be obtained from a variety ofsources including cord blood, bone marrow, and mobilized peripheralblood. Purification of CD34+cells can be accomplished by antibodyaffinity procedures (see Ho et al., Stem Cells 13 (suppl. 3): 100-105(1995); see also Brenner, J. Hematotherapy 2: 7-17 (1993)). Cells canalso be isolated and cultured from patients. Alternatively, the cellsused for ex vivo procedures can be those stored in a cell bank (e.g., ablood bank). The advantage to using stem cells is that they can bedifferentiated into other cell types in vitro, or can be introduced intoa mammal (such as the donor of the cells) where they will engraft in thebone marrow. Methods for differentiating bone marrow cells in vitro intoclinically important immune cell types using cytokines such as GM-CSF,IFN-γ and TNF-α are known (see, e.g., Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Delivery of a nucleic acid can also be achieved using in vivo genetherapy. The lipid:nucleic acid complexes of the invention can beadministered directly to a patient, preferably a human. In vivo and exvivo administration is by any of the routes normally used forintroducing a molecule or cell into ultimate contact with blood ortissue cells. Lipid:nucleic acid complexes of the invention areadministered in any suitable manner, preferably with pharmaceuticallyacceptable carriers.

Suitable methods of administering such non-viral particles in thecontext of the present invention to a patient are known to those skilledin the art. Preferably, the pharmaceutical compositions are administeredusing aerosol administration (e.g., using a nebulizer or otheraerosolization device), and parenterally, i.e., intra-arterially,intravenously, intraperitoneally, subcutaneously, or intramuscularly.More preferably, the pharmaceutical compositions are administered viaaerosol administration or intravenously or intraperitoneally by a bolusinjection. Particular formulations which are suitable for this use arefound in Remington's Pharmaceutical Sciences (17th ed. 1985). Typically,the formulations will comprise a solution of the lipid:nucleic acidcomplexes suspended in an acceptable carrier, preferably an aqueouscarrier.

V. Pharmaceutical compositions

Pharmaceutical compositions comprising the lipid:nucleic acid complexesof the invention are prepared according to standard techniques andfurther comprise a pharmaceutically acceptable carrier. Generally,normal saline will be employed as the pharmaceutically acceptablecarrier. Other suitable carriers include, e.g., water, buffered water,isotonic solution (e.g., dextrose), 0.4% saline, 0.3% glycine, and thelike, including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. These compositions may be sterilized byconventional, well known sterilization techniques. The resulting aqueoussolutions may be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the lipid:nucleic acid complex suspensionmay include lipid-protective agents which protect lipids againstfree-radical and lipid-peroxidative damages on storage. Lipophilicfree-radical quenchers, such as alphatocopherol and water-solubleiron-specific chelators, such as ferrioxamine, are suitable.

The concentration of lipid:nucleic acid complexes in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.05%, usuallyat or at least about 2-5% to as much as 10 to 30% by weight and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, immunolipid:nucleic acid complexes composedof irritating lipids may be diluted to low concentrations to lesseninflammation at the site of administration. The amount of lipid:nucleicacid complex administered will depend upon the particular Fab′ used, thedisease state being treated, and the judgement of the clinician.Generally the amount of lipid:nucleic acid complexes administered willbe sufficient to deliver a therapeutically effective dose of the nucleicacid. The quantity of lipid:nucleic acid complex necessary to deliver atherapeutically effective dose can be determined by one skilled in theart. Typical lipid:nucleic acid complex dosages will generally bebetween about 0.01 and about 50 mg nucleic acid per kilogram of bodyweight, preferably between about 0.1 and about 10 mg nucleic acid/kgbody weight, and most preferably between about 2.0 and about 5.0 mgnucleic acid/kg of body weight. For administration to mice, the dose istypically 50-100 μg per 20 g mouse.

VI. Assaying blood half-life

One aid for lipid:nucleic acid complex localization in a target tissueis an extended lipid:nucleic acid complex lifetime in the bloodstreamfollowing administration. One measure of lipid:nucleic acid complexlifetime in the bloodstream is the blood/RES ratio determined at aselected time after complex administration. Typically lipid:nucleic acidcomplexes containing a label (e.g., fluorescent marker, electron densereagent, or radioactive marker), either internal in the complex or boundto a lipid comprising the complex are injected into the test organism. Afixed period of time later, the organism is sacrificed and the amount oflabel detected in the blood (e.g., by measuring luminescence, orscintillation counting) is compared to that localized in particulartissues (e.g., liver or spleen).

The time course of retention of lipid:nucleic acid complexes in theblood may also simply be determined by sampling blood at fixed intervalsafter administration of label-containing lipid:nucleic acid complexesand determining the amount of label remaining in the circulation. Theresult may be expressed as the fraction of the original dose.

VII. Assaying tissue transfection by the lipid:nucleic acid complexes

Transfection of target cells by the lipid:nucleic acid complexes of thisinvention may similarly be determined by administering lipid:nucleicacid complexes containing a nucleic acid that is itself detectable orthat encodes a detectable product. Biological samples (e.g., tissuebiopsies or fluid samples) are then collected and assayed fortransfection by detecting the presence of the transfected nucleic aciditself or by detecting the presence of the expressed product of thenucleic acid.

The nucleic acid itself can be selected to have a sequence that isreadily detectable, e.g., by nucleic acid amplification. In thisinstance, the nucleic acid would be selected that has primer sitesselected so as to permit unique amplification of the subject nucleicacid and no other in the sample of the biological tissue that is to beassayed for transfection.

Means for detecting specific DNA sequences are well known to those ofskill in the art. For instance, oligonucleotide probes chosen to becomplementary to a select subsequence with the region can be used.Alternatively, sequences or subsequences may be amplified by a varietyof DNA amplification techniques including, but not limited to polymerasechain reaction (PCR) (Innis et al., PCR Protocols: A guide to Methodsand Application (1990)), ligase chain reaction (LCR) (see Wu & Wallace,Genomics 4: 560 (1989); Landegren et al., Science 241: 1077 (1988);Barringer et al., Gene 89: 117 (1990), transcription amplification (Kwohet al., Proc. Natl. Acad. Sci. USA 86: 1173 (1989)), and self-sustainedsequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA 87:1874 (1990)).

In a particularly preferred embodiment, transfection is evaluated bydetecting the presence or absence or quantifying a gene product in oneor more tissues. Any gene that expresses an easily assayable productwill provide a suitable indicator for the present assay. Suitablereporter genes are well known to those of skill in the art. Theyinclude, but are not limited to, bacterial chloramphenicol acetyltransferase (CAT), beta-galactosidase, or luciferase (see, e.g., Alam etal., Analytical Biochemistry 188: 245-254 (1990)). One particularlypreferred reporter gene is the Fflux gene as illustrated in theExamples.

VIII. Assaying shelf life

As indicated above, the term “shelf life” is used herein to refer to theperiod of time the lipid:nucleic acid complex can be stored (underdefined conditions e.g., in a buffer at 4° C.) before losing itsbiological activity. The biological activity assayed for determinationof shelf life in the present invention is the ability of thelipid:nucleic acid complex to transfect mammalian cells in vivo afterintravenous administration.

In a preferred embodiment the shelf life is determined by storing thelipid:nucleic acid complexes for varying periods of time, injecting oneor more test animals with the complex and assaying selected tissues inthe animal for transfection (e.g., expression of a reporter gene) asdescribed above and as illustrated in the examples.

It will be appreciated that shelf life can be expressed in absoluteterms, i.e., the length of time the composition can be stored beforelosing activity. Alternatively, shelf life can be expressed in relativeterms by reference to a different composition. Thus, for example, whenthe subject complex shows transfection activity after a fixed period ofstorage and this activity is greater than the activity of a differentcomplex similarly stored for the same amount of time, the subjectcomplex is said to have an increased shelf life as compared to thedifferent complex.

IX. Targeting lipid:nucleic acid complexes to specific tissues

Specific targeting moieties can be used with the lipid:nucleic acidcomplexes of the invention to target specific cells or tissues. In oneembodiment, the targeting moiety, such as an antibody or antibodyfragment, is attached to a hydrophilic polymer and is combined with thelipid:nucleic acid complex after complex formation. Thus, the use of atargeting moiety in combination with a generic effector lipid:nucleicacid complex provides the ability to conveniently customize the complexfor delivery to specific cells and tissues.

Examples of effectors in lipid:nucleic acid complexes include nucleicacids encoding cytotoxins (e.g., diphtheria toxin (DT), Pseudomonasexotoxin A (PE), pertussis toxin (PT), and the pertussis adenylatecyclase (CYA)), antisense nucleic acid, ribozymes, labeled nucleicacids, and nucleic acids encoding tumor suppressor genes such as p53,p110Rb, and p72. These effectors can be specifically targeted to cellssuch as cancer cells, immune cells (e.g., B and T cells), and otherdesired cellular targets with a targeting moiety. For example, asdescribed above, many cancers are characterized by overexpression ofcell surface markers such as HER2, which is expressed in breast cancercells, or IL17R, which is expressed in gliomas. Targeting moieties suchas anti-HER2 and anti-IL17R antibodies or antibody fragments are used todeliver the lipid:nucleic acid complex to the cell of choice. Theeffector molecule is thus delivered to the specific cell type, providinga useful and specific therapeutic treatment.

X. Lipid:nucleic acid complex kits

The present invention also provides for kits for preparing theabove-described lipid:nucleic acid complexes. Such kits can be preparedfrom readily available materials and reagents, as described above. Forexample, such kits can comprise any one or more of the followingmaterials: liposomes, nucleic acid (condensed or uncondensed),hydrophilic polymers, hydrophilic polymers derivatized with targetingmoieties such as Fab′ fragments, and instructions. A wide variety ofkits and components can be prepared according to the present invention,depending upon the intended user of the kit and the particular needs ofthe user. For example, the kit may contain any one of a number oftargeting moieties for targeting the complex to a specific cell type, asdescribed above.

The kit may optionally include instructional materials containingdirections (i.e., protocols) providing for the use of the cationiclipid:nucleic acid complex for transfecting cells in vivo, ex vivo, orin vitro. Typically, the instruction materials describe the procedurefor preparing the lipid:nucleic acid complex from liposomes and nucleicacid, as described above. The instruction materials also describe how tomix the hydrophilic polymer with the lipid:nucleic acid complex.Additionally, the instruction materials can describe procedures fortransfecting cells with the lipid:nucleic acid complex.

While the instructional materials typically comprise written or printedmaterials, they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

XI. Preparation of lipidic microparticles with surface attached proteins

A. General

The present invention also provides for the preparation of lipidicmicroparticles with surface-attached proteins. As noted in theBackground section, above, the art teaches that soluble proteins such asantibodies are so large that their tendency to solubilize overwhelms thetendency of a hydrophobic domain of a linker molecule attached to theprotein to become stably associated with a lipidic microparticle. Theteaching has therefore been that, while small peptides conjugated to alinker molecule of approximately the same size or larger might permitthe hydrophobic domain of a linker molecule to become stably associatedwith a lipidic microparticle, proteins conjugated to a linker molecule,which is much smaller than the protein, would not be able to do so.

We have now found that, contrary to the teachings of the art, proteinsmany times larger than a linker molecule can be conjugated to a linkermolecule and still be successfully and stably attached to a lipidicmicroparticle. The Examples, below, demonstrate that proteins many timeslarger than the linker molecules to which they are conjugated can besuccessfully attached to lipidic microparticles. This discovery expandsthe types of agents with which such microparticles can be loaded.Further, it expands the range of methods by which such microparticlescan be made and still attached to proteins, since the attachment can nowtake place under conditions where the stability of the microparticle,such as a liposome, will not be jeopardized.

Preferably, the proteins used in this method have a molecular weightbetween about 6,000 and about 1,000,000 Daltons. More preferably, theproteins have a molecular weight between about 10,000 and about 600,000Daltons. Even more preferably, the proteins have a molecular weightbetween about 15,000 and about 250,000 Daltons. Most preferably, theproteins have a molecular weight between about 20,000 and about 75,000Daltons

B. Attachment of proteins by incubating lipidic microparticles withproteins conjugated to linker molecules

For use in this invention, a protein can first be conjugated to a linkermolecule comprising (a) a hydrophobic domain, (b) a hydrophilic polymerchain terminally attached to the hydrophobic domain, and (c) a chemicalgroup reactive to one or more functional groups on a protein moleculeand attached to the hydrophilic polymer chain at, or near to, theterminus contralateral to the hydrophobic domain. Such linker moleculesare known in the art (Allen & Martin, U.S. Pat. No. 5,527,528; Shahinian& Sylvius, Biochim. Biophys. Acta, 1239:157-167 (1995); Zalipsky et al.,J. Controlled Release 39:153-161, 1996; Kirpotin et al., Biochemistry,36:66-75 (1997)).

The hydrophobic domain of the linker molecule may be, for example, adiacylglycerol, a phospholipid, a sterol, such as cholesterol, or adiacylamide derivative, such as N,N-distearoyl-glycineamide. Thehydrophilic polymer chain may be, for example, poly(ethylene glycol),polyglycidol, poly(vinyl alcohol), poly(vinyl pyrrolidone),polyoxazolidinone, polysaccharide, or a copolymer which includes theblocks of the above polymers. The chemical reactive group may be, forexample, an amino group, carboxy group, thiol group, malemido group,iodoacetamido group, vinylsulfone group, aldehyde group, hydrazinegroup, ketone group, cyanure chloride group, or any other functionalgroup known in the art to form linkages with proteins. A protein may bean antibody, an enzyme, a growth factor, a hormone, a nucleicacid-binding protein, or any other protein of utility for a particularintended application.

In a preferred embodiment of the invention, the protein is an Fab″fragment of an antibody, or a single chain antibody. In a furtherpreferred embodiment, the single chain antibody is a Fv antibodyproduced through selection from a phage display library. Maleimidogroups, which react with cysteine residues in the protein, are preferredas the reactive group for use with an Fab′ antibody fragment or singlechain antibody.

The conjugation of the protein to the linker can be effected by any of anumber of methods known in the art for protein conjugation. In apreferred method, the linker can be simply dissolved in aqueous buffer(which is possible due to the presence of the hydrophilic polymerdomain) and incubated with the protein of choice to afford formation ofa stable bond between the chemical reactive group of the linker and theappropriate functional group of the protein. The conjugate can befurther purified from the excessive linker and any unconjugated proteinby salting-out, dialysis, chromatography and other methods known in theart of protein purification. Alternatively, the conjugate can be usedwithout further purification.

Conjugated protein is then incubated with the lipidic microparticles inan aqueous medium for a time sufficient for the hydrophobic domain ofthe conjugate to merge into the surface lipid layer of the particle. Thetime required will depend on the lipid composition of the microparticle,the nature of the hydrophobic domain, and the temperature of incubation.Typically, the time of incubation will lie in the range from about 1minute to about 50 hours. The time necessary for incubation willdecrease as the temperature at which the incubation is conducted isincreased. Thus, at 37° C., the incubation will generally takeovernight, while at 55-60° C., the incubation will generally take 5-60minutes, with 15-30 minutes being preferred. Incubation timesappropriate for any particular combination of microparticle, hydrophobicdomain, and temperature, can be determined using the assays taught inthe Examples, below.

C. Preparation of proteins containing hydrophobic domains which willself-insert into a lipidic microparticle

In an alternative embodiment, a hydrophobic anchor and a hydrophilicpolymer chain are introduced into a protein molecule by recombinant DNAand protein engineering methods. In this case, a hydrophilic polymericdomain, as described above, is introduced into the protein of interestby a terminally appended polyaminoacid sequence containing primarilyamino acids with hydrophilic side chains. A hydrophobic anchor isintroduced into the construct during its biosynthesis via a lipidmodification site positioned at the distal end of the terminallyappended polyaminoacid sequence.

EXAMPLES

The invention is illustrated by the following examples. These examplesare offered to illustrate, but not to limit the present invention.

Example 1

Preparation of stable lipid:plasmid DNA complexes for in vivo genedelivery

A. Materials and methods

1. Lipids & other reagents

DOPE was purchased from Avanti (Alabaster, Ala.). Highly purifiedCholesterol was obtained from Calbiochem (San Diego, Calif.). DDAB anddextran (M.W. 40,000) were purchased from Sigma (St. Louis, Mo.). DDABwas recrystalized once from acetone-methanol solution. D-luciferin wasobtained from Boehringer Mannheim. PEG-PE was a gift from SequusPharmaceuticals (Menlo Park, Calif.). DC-Chol, MMCE and DOGS wereobtained from UCSF Gene Transfer Vehicle Core of Gene Therapy Center.ESPM, DOTAP, POEPC, DOEPC, DMEPC and DODAP were gifts from Avanti(Alabaster, Ala.). Chloroform solution of each lipid was stored underargon in sealed ampules at −40° C. Other reagents of possible highestgrade were purchased and used without further purification.

2. Preparation of liposomes

Small cationic liposomes were prepared in 5% (w/v) dextrose solution inthe following fashion. DDAB or other cationic lipids in chloroform wasmixed with DOPE or/and cholesterol in a desired molar ratio, and thesolvent was removed slowly under reduced pressure at 50° C. on a rotaryevaporator. The dry lipid film was hydrated with 5% dextrose solutionprewarmed to 50° C. and the container was sealed under argon. Thehydrated lipid suspension was sonicated in a bath sonicator (LabSupplies, Hicksville, N.Y.) for 5-10 min at 50° C. The finalconcentration of liposomes was 5 mM cationic lipid and the size ofliposomes was measured by dynamic light scattering to be 195±65 nm.Sonicated liposomes were stored under argon at 4° C. until use.

3. Luciferase reporter system

Plasmid, pCMV/IVS-luc⁺, was constructed as follows. A fragmentcontaining the CMV promoter and synthetic IgE intron was excised frompBGt2.CAT using Spe I and Hind III and cloned into pBSIIKS⁺. The cDNAencoding the modified firefly luciferase (luc+) including SV40 late poly(A) signal was cut from the pGL3-Basic Vector (Promega) with Hind IIIand Sal I and was put into the pBS-CMV-IVS clone downstream of thesplice. Plasmids were purified using alkaline lysis procedures adoptedand devised by Qiagen Corp. (Chatsworth, Calif.). Plasmid purity wasmeasured by the ratio of absorbance at 260 nm vs 280 nm, and stored inbuffer containing 10 mM Tris-Cl and lmM EDTA at pH 8.0 at concentrationsof 1-2 mg/ml.

4. Preparation of transfection complexes

Prior to the transfection experiments, the optimal DNA/liposome ratiofor forming complexes which were not large aggregates was determined bymixing fixed amount plasmid to various amount of liposomes. In general,the transfection complexes were formed by pipetting plasmid intoliposome suspension of equal volume and mixing rapidly. Routinely,liposomes containing 8-12 nmole of DDAB could complex with 1 μg plasmidwithout forming visible large aggregates. Such complexes have excesspositive charge, but still tend to aggregate with time during storage at4° C. and lose transfection activity in 4 days. For in vitroexperiments, which called for much dilute complexes, cationiclipid:plasmid DNA complexes (“CLDC”) at 5 nmole DDAB per μg DNA wereused. To keep the lipid:plasmid DNA complexes from forming largeaggregates and losing transfecting activity with time, two approacheswere taken: (1) incorporating a small amount of PEG-PE (approx. 1% moleratio) into lipid:plasmid DNA complexes within a few minutes after theirpreparation; and/or (2) condensing plasmid with polyamines (e.g., 0.05to 5.0 nmole of spermidine per μg DNA) prior to mixing with liposomes.The optimal amount of the polyamines was determined by titratingpolyamines to DNA before forming large aggregates. The size of thesecomplexes was estimated by dynamic light scattering to be in the rangeof 410±150 nm.

5. Assay of reporter gene expression

Purified luciferase was purchased from Boehringer Mannheim as a standardfor calibrating the luminometer and constructing a control standard forthe relative specific activity of luciferase. Reporter gene expressionin a tissue extract was presented in nanogram quantities by convertingrelative light unit measured from a luminometer into weight unitaccording to a standard curve. Luciferase expressed in cells or tissueswas extracted with chemical cell lysis. Effective lysis buffer consistedof 0.1 M potassium phosphate buffer at pH 7.8, 1% Triton X-100, 1 mM DTTand 2 mM EDTA.

Female CD1 mice (4-6 weeks old, weighing approx. 25 g) were obtainedfrom Charles River Laboratory. Mice received lipid:plasmid DNA complexesby tail vein injection and were sacrificed 24 h later. The anesthetizedanimals were perfused with cold phosphate-buffered saline (PBS) viaheart puncture. Each tissue was dissected and washed in PBS, and thenhomogenized in 6 ml round-bottomed culture tube containing 500 μl oflysis buffer. The samples were kept at room temperature for 20 min withoccasional mixing. The homogenized samples were centrifuged for 10 minat 3000 rpm in an Eppendorf centrifuge. Luciferase activity of eachtissue was measured by mixing 100 Al of the reconstituted luciferasesubstrate (Promega, Madison, Wis.) with 20 μl of the supernatant oftissue homogenate in the injection system of a luminometer. Peak lightemission was measured for 10 sec. at 20° C. Relative light units of eachsample were converted to the amount of luciferase in the tissue extractby comparing with a standard curve which was established for each set ofexperiments. The protein content of the extract was determined usingprotein assay kits (BioRad, Richmond, Calif.). Background was the countof lysis buffer only.

SK-BR-3 cells (Park et al., Proc. Natl. Acad. Sci. USA 92: 1327-1331(1995)) were cultured in McCoy's 5 A medium supplemented with 10%heat-inactivated bovine calf serum and in 5% CO₂. SK-BR-3 cells inmonolayer culture were plated at 50,000 cells per well in 12-well platesand incubated overnight. Each well received 0.5˜1 μg of pCMV/IVS-luc⁺within 20 min of complex formation. Cells were harvested after 24 hr ofincubation with complexes at 37° C. Luciferase activity in cells wasdetermined as described above.

B. Results

1. Optimizing the “helper” lipid

The use of cationic liposomes for in vitro gene transfer has becomewidespread since Felgner et al. published their study (Felgner et al.,Proc. Natl. Acad. Sci. USA 84: 7413-17 (1987)). It was established later(Felgner et al., J. Biol. Chem. 269: 2550-2561 (1994)) that DOPE is byfar the most efficient “helper” lipid for in vitro gene transfection andthis result has confirmed by several laboratories (Farhood et al., inGene Therapy for Neoplastic Diseases, pp 23-55 (Huber & Lazo eds.,1994); Zhou et al., Biochim. Biophys. Acta 1189: 195-203 (1994)). It hasbeen suggested, on the basis of in vitro studies, that DOPE mayfacilitate the cytoplasmic delivery via membrane fusion once positivelycharged lipid:plasmid DNA complexes are bound to the cell membrane (Zhouet al., Biochim. Biophys. Acta 1189: 195-203 (1994)). Even though Friendet al. did not obtain any morphological evidence that the DOTMA/DOPElipid:plasmid DNA complexes fuse directly with the plasma membrane, theydo not exclude the possibility of fusion events (Friend et al., Biochim.Biophys. Acta 1278: 41-50 (1996)). They suggested that the complexes areendocytosed and the cationic lipids disrupt the endosomal/lysosomalmembranes and then facilitate an escape of the DNA complexes into thecytoplasm and eventually into the nucleus.

Contrary to most expectations, the “helper” role of DOPE establishedfrom in vitro studies is not evident for in vivo gene delivery followingi.v. injection of the complexes. When DOPE was included in DDAB cationicliposomes, the in vivo gene transfection was inhibited. ThisDOPE-dependent inhibition is shown in FIG. 1. Cholesterol, not DOPE, wasfound to be effective as “helper” lipid for in vivo gene delivery. Therewas a ten-fold reduction in luciferase expression in mouse lungs whenhalf of the cholesterol was replaced with DOPE. The in vivo results ofDDAB and other cationic liposomes are not consistent with the generalassumption that DOPE is a suitable “helper” lipid. On the contrary, DOPEin cationic lipid:plasmid DNA complexes attenuates the in vivotransfection to such a great degree that DOPE is considered as aninhibitory agent in formulations for in vivo gene delivery. Cholesterolhas been chosen for in vivo studies in recent published reports (Liu etal., J. Biol. Chem. 270: 24864-70 (1995); Solodin et al., Biochemistry34: 13537-44 (1995)) in which the authors do not elaborate on how andwhy they selected different “helper” lipids for their experimentaldesigns, i.e. DOPE for in vitro and cholesterol for in vivo studies.Stabilization of anionic and neutral liposomes in blood by cholesterolhas been known for a long time (Mayhew et al., Cancer Treat. Rep. 63:1923-1928 )1979)). It is therefore obvious that for systemic genedelivery, one has to consider the stability of lipid:plasmid DNAcomplexes in blood, various components of which are known to react withmacromolecular complexes. In fact, the preliminary study of variousformulations of lipid:plasmid DNA complexes using freeze-fractureelectron microscopy has shown that the cholesterol-containing complexeswere structurally more stable than the DOPE-containing complexes in thepresence of serum.

Using DDAB/Chol lipid:plasmid DNA complexes (8 nmole DDAB/μg DNA) for invivo transfection experiments, detectable luciferase expression in thelung of 25 g mouse required a DNA dose ranging from 30 μg to 60 μg.Routinely 40˜60 μg plasmid DNA per mouse gave consistent geneexpression. The amount of DDAB usually associated with 80 μg DNA (ormore) per mouse was found to be too toxic to the animal. The expressionof luciferase in various tissues is shown in FIG. 2. As observed before(Zhu et al., Science 261: 209-211 (1993); Liu et al., J. Biol, Chem.270: 24864-70 (1995); Solodin et al., Biochemistry 34: 13537-44 (1995)),maximal expression was found in lung tissue. For 60 μg plasmid injected,1-2 μg luciferase per mg tissue protein was routinely obtained. FIG. 3shows the duration of reporter gene expression in lung tissue.Expression of luciferase decreased rapidly and reached undetectablelevels in 2 weeks. Zhu et al. reported that following i.v. injection ofDOTMA/DOPE (1:1)—plasmid complexes into adult mice, the expression ofthe reporter gene (CAT) is widespread among various tissues and themaximum expression is from complexes with a ratio of 1 I μg plasmid to 8nmole total lipids (Zhu et al., Science 261: 209-211 (1993)). However,at this ratio (corresponding to 1 μg plasmid to 4 nmole cationic lipid),DDAB/Chol lipid:plasmid DNA complexes tended to aggregate and did notproduce measurable gene expression in this investigation.

Since different reporter genes have been used among differentlaboratories, it has been difficult to attribute the variations in theefficiency of in vivo gene delivery to changes in the formulation ofliposomes. For a direct comparison of the results in the literature, therelative light units of luciferase activity measured from a luminometerwas converted to a standard of purified luciferase. By doing so, thepeak transfection activity of DDAB/Chol formulations was 3 orders ofmagnitude higher than values reported recently in comparable experiments(Thierry et al., Proc. Natl. Acad. Sci. USA 92: 9742-9746 (1995)). Giventhat same reporter gene along with same promoter in the experimentaldesign, the difference in expression may reflect the selection ofliposome formulation. In fact, DDAB/Chol was one of the most efficientgene delivery vehicle among many formulations from 18 different cationiclipids which was screened recently. Preliminary results of expression inmouse lung following i.v. injection indicated that DOTMA/Chol,DOTAP/Chol, MMCE/Chol and ESPM/Chol gave 10-100% transfection activityof DDAB/Chol, DOGS/Chol, POEPC/Chol, LYSPE/DOPE and DC-Chol/DOPE gave1-10% of DDAB/Chol. DOEPC/Chol, DMEPC/Chol, DODAP/Chol and DDAB/DOPE didnot give any measurable activity.

In parallel with the transfection studies, the morphology of thesecomplexes in serum and in cell medium was examined by freeze-fractureelectron microscopy. When examined in 50% mouse serum (10 minuteincubation time), non-stabilized, one day old CLDC are as small as theyare in buffer at low ionic strength (100-250 nm) but show very fewprotrusions. Six day old, non stabilized CLDC incubated in 50% mouseserum appeared as densely packed aggregates of spherical particles, witha high number of attached particles. Such formulations have lost all oftheir in vivo transfection activity within 4 days. Residual fibrillarprotrusions are not observed.

PEG-PE stabilized CLDC incubated in 50% mouse serum were small (100-200nm) even at six days. Similarly, CLDC prepared with condensed DNA werealso quite small even after six days of storage. Specifically, the CLDCwere shaped like “map pins” that were structurally stable in thepresence of serum.

After incubation in cell medium (RPMI-1640 with 10% FCS), non-stabilizedsix day old CLDC were morphologically similar to those incubated inmouse serum, as described above. These complexes, however, were moreloosely packed and showed no fibrillar protrusions. Similar morphologywas observed with PEG-PE stabilized CLDC and condensed DNA CLDCincubated in cell medium.

2. Increasing shelf life for transfection activity

The relationship between structural stability and transfection activityof lipid:plasmid DNA complexes has not been detailed in the publishedreports so far. Screening procedures have been established to avoidlarge aggregates of lipid:plasmid DNA complexes by changing the ratio ofDNA to lipid from net negatively charged to positively charged.Lipid:plasmid DNA complexes of each particular cationic lipid at variousratios of DNA/lipid were prepared and the resulting stable andmetastable formulations were used for in vivo transfection. Complexeswhich contained 8 to 12 nmole of cationic lipid per Ag DNA were found tohave the highest in vivo transfection activity. However, thetransfection activity of these complexes decreased with time. Withoutmodifying the procedures of forming the lipid:plasmid DNA complexes,there was a visible aggregation within a few days, and the transfectionactivity decreased by more than a thousand fold to almost backgroundlevels after one month's storage at 4° C. (FIG. 4). Therefore,formulation of stabilized lipid:plasmid DNA complexes was undertaken,which could maintain high in vivo transfection activity during storage.

i. Increasing transfection stability: PEG-PE

Inserting PEG-PE (1% of total lipid) into the freshly formedlipid:plasmid DNA complexes not only could prevent the complexes fromaggregating during storage, but the PEG-PE containing complexes alsoexhibited reasonably high transfection activity in vivo, only slightlylower activity as compared to the complexes without PEG-PE (FIG. 4).Incorporation of PEG-PE into the complexes is evident in view of thedose-related inhibition of the transfection activity with increasingpercentage of PEG-PE (results not shown). Unexpectedly, storage of thecomplexes containing PEG-PE at 4° C. slyly restored the originalactivity, as shown in FIG. 4. The mechanistic aspects of the inhibitioneffect on transfection by PEG-PE, as well as the recovery of theactivity following storage at low temperature, are not known at presenttime.

ii. Increasing transfection stability: polyamines

In addition to the role of PEG-PE in increasing the shelf life oflipid:nucleic acid complexes, condensing nucleic acid with polyaminesalso gave a similar unexpected increase in shelf life of the complexes.The lipid:plasmid DNA complexes formed with condensed DNA were stable ata lower ratio of lipid to DNA without aggregation. FIG. 4 shows thelevel of in vivo transfection activity of such preparation, and its fateduring storage. Again, an unexpected increase of the transfectionactivity was found in aged polyamine-treated lipid:plasmid DNAcomplexes, when compared to that of the samples which were notpretreated with polyamines and used immediately after complexes wereformed. A different approach to obtain stable cationic lipid/DNAcomplexes by complexing plasmid with lipid in lipid-detergent micelleswas published recently (Hofland et al., Proc. Natl. Acad. Sci. USA 93:7305-7309 (1996)). However, only 30% of the transfection efficiency wasmaintained by such complexes in 15% serum for in vitro, and no in vivoresults were reported.

iii. Increasing transfection stability: lyophilization

Finally, conditions have been established for the stabilization oflipid:plasmid DNA complexes by lyophilization. Liposomes composed ofDDAB/Chol suspended by sonication in 5% (w/v) of dextran in water, whenmixed with DNA in 1:10 ratio (μg DNA per nmole DDAB) as described inmethods, could be lyophilized without loss of activity. The finalconcentration of dextran in which lipid:plasmid DNA complexes wereformed was 8% (w/v). The lyophilized preparations were reconstituted byadding distilled water and their transfection activity in the lungs ofmice after i.v. injection was measured by luciferase reporter geneexpression. Freezing and thawing the reconstituted preparation did notaffect the activity (usually 1-2 ng luciferase protein per mg tissueprotein).

Several of the cationic lipid:plasmid DNA complexes described herein arestable and can give consistent in vivo transfection activity (rangingfrom 0.5 to 2 ng luciferase per mg tissue protein) even after longstorage at 4° C. or lyophilization. Formulations containing cholesterolas the “helper” lipid generate much higher in vivo transfectionefficiency. Stabilizing the complex structure by PEG-PE maintains thecomplex activity in storage and may prolong the circulation time inblood for targeting to specific tissues. Condensing the DNA withpolyamines before lipid complexation enhances in vitro storage andlevels of activity in vivo. The methodical approach for producing stableformulations of lipid:plasmid DNA complexes exhibiting high transfectionactivity in vivo confers advantages for establishing pharmaceuticallyacceptable preparations, and therefore facilitates liposome based genetherapy.

Example 2

In vitro transfection of lipid:plasmid DNA complexes with targetingligands

A. Preparation of Fab′ fragments

Cloned rhuMAbHER2 sequences for heavy and light chain were co-expressedin E. coli as previously described (Carter et al., Biotechnology 10:163-167 (1992)). The antibody fragment, rhuMAbHER2-Fab′, was recoveredfrom E. coli fermentation pastes by affinity chromatography withStreptococcal protein G (Carter et al., Biotechnology 10:163-167(1992)), typically yielding Fab′ with 60-90% containing reduced freethiol (Fab′-SH).

B. Preparation of liposomes

Condensed DNA was complexed with three different lipid compositions,using the methods described above in Example 1, with the followingmodifications. The first complex was made with DDAB/DOPE (1/1), whichproduced cationic liposomes complexed with DNA only, as described above.The second complex was made with DDAB/DOPE (1/1) with 1% PEG-PEderivatized with maleimide at the ultimate position of PEG, producingCLDC with the steric stabilization component added after complexationwith the DNA. The third complex was made with DDAB/DOPE (1/1) with 1%PEG-PE derivatized with the Fab′ fragment of a humanized anti-Her-2antibody attached to the ultimate position of PEG via the free thiolgroup to the maleimide residue. This produced CLDC with the targetingligand attached to the steric stabilization component added after thecomplexation with the DNA.

C. Transfection and results

Cells were transfected as described above in Example 1, but withoutstorage of the lipid:plasmid DNA complex. Two cell lines were used inthis Example. The first cell line was MCF-7; cells of this cell line donot overexpress the HER-2 receptor. These cells were cultured in DMEH-21 with 10% bovine calf serum and in 5% CO₂. The second cell line wasSK-BR3 cells, cells of which overexpress the HER-2 receptor, cultured inMcCoy's 5A medium with bovine calf serum in 5% CO₂. In both cases, thecells (˜5×10⁴ cells per well) were transfected and incubated with 12 μgplasmid DNA complexed with lipid as described above (PCMV/IVS-luc+,luciferase reporter gene described above) for 4 hours at 37° C. Thesupernatant was then aspirated, fresh medium was added and the cellswere incubated for 24 hours at 37° C. Cells were then harvested bywashing with PBS (Ca/Mg free) and then suspended in lysis buffer for theluciferase assay, as described above.

FIG. 5A shows that transfection of non-target cells, not over-expressingthe HER-2 receptor, was inhibited by the addition of PEG-PE, even in thepresence of the targeting ligand conjugated at the tip of PEG via theterminal maleimide residue. FIG. 5B shows that transfection of targetcells overexpressing the HER-2 receptor was also inhibited by theaddition of PEG-PE, but the transfection activity was restored andaugmented when the PEG-PE was conjugated to a targeting ligand, whichrecognizes the HER-2 receptor.

Comparison of FIGS. 5A and 5B indicates that the targeted immuno-CLDCwere active in transfecting target cells much more efficiently thannon-target cells. This result occurs because the addition of theligand-carrying stabilizing agent (PEG-PE) conjugated toanti-HER-2-Fab′), which inhibits the transfection of non-target cells(FIG. 5A) but augments transfection of the target cells (FIG. 5B).

Example 3

Preparation of the linkermaleimido-propionylantido-PEG2000-distearoylphosphatidylethanolamine(Mal-PEG-DSPE).

100 mg (44 mol) of ω-maleimidopropionylamido-poly(ethyleneglycol)-α-succinimidylcarbonate (Mal-PEG-NHS; Shearwater Polymers, Inc.)prepared from poly(ethylene glycol) (molecular weight 2,000), 33 mg (44μmol) of distearoyl-phophatidylethanolamine (DSPE; Avanti Polar Lipids),and 12 ml (86 μmol) of triethylamine in 1 ml of chloroform, wereincubated for 6 hours at 45° C. At this time, thin layer chromatographyon silica (solvent, chloroform/methanol 7:3) indicated completeconversion of DSPE into faster moving, ninhydrin-negative productidentified as Mal-PEG-DSPE. This product was purified by columnchromatography on silica, using stepwise gradient of methanol inchloroform (5%, 10%, 15% of methanol by volume). Pure Mal-PEG-DSPE waseluted at 15% methanol. Yield, 85 mg (67% of theory). R_(f) 0.27-0.29(Silica 60, CHCl₃—MeOH—H₂O 65:25:4). Ratio of maleimido groups tophosphate, 0.95-1.02.

Alternatively, this linker may be prepared as described in U.S. Pat. No.5,527,528 or in Kirpotin et al. (Biochemistry, 36:66-75 (1997)).

Example 4

Conjugation of Mal-PEG-DSPE with Fab′ fragment of an antibody reactiveagainst HER2 oncoprotein.

300 nmol of Mal-PEG-DSPE in 0.5 ml of chloroform were placed in a glasstest-tube and the solvent was removed in vacuum. The dry residue wasdissolved in 1 ml of MES-20 buffer (20 mM morpholinoethane sulfonicacid, 144 mM sodium chloride, 2 mM ethylenediamine tetraacetic acid, andNAOH to pH 6.0). 2,5 ml of solution containing 0.57 mg/ml of Fab′fragments of a recombinant humanized monoclonal antibody againstextracellular domain of HER2 oncoprotein (rhuMAbHER2, Genentech, Inc.)was added to the Mal-PEG-DSPE solution, and the pH was carefullyadjusted to 7.2-7.4 with diluted NaOH. The mixture was incubated underargon at room temperature for 2.5 hours, and the reaction was stopped byaddition of 0.2 M cysteine hydrochloride to a final concentration of 5mM. Fifteen minutes after the addition of cysteine, the reaction mixturewas dialyzed against HEPES-buffered saline (20 mM hyrdoxyethylpiperazinoethanesulfonic acid, 144 mM NaCl, NaOH to pH 7.2), concentrated byultrafiltration through a YM-10 membrane (Amicon) under pressure, andsterilized by filtration through a 0.2 μm cellulose acetate filter. Thereaction products were analyzed by polyacrylamide gel electrophoresis inthe presence of sodium dodecyl sulfate (SDS-PAGE), with Coomassie Bluestaining. Total protein was determined the dye binding assay (Bio-Rad).The assay revealed 62% conversion of the original protein (M.w. 46,000)into slower-moving product (M.w. 49,000) consistent with the expectedconjugate. Total protein recovery in the products was 98%.

Example 5

Conjugation of Mal-PEG-DSPE with a single chain Fv antibody reactiveagainst HER2 oncoprotein.

150 nmol of Mal-PEG-DSPE were dissolved in 0.5 ml of MES-20 and reactedwith 0.5 ml of solution containing 0.7 mg/nml of single-chain Fvantibody C6.5Cys reactive against extracellular domain of HER2oncoprotein. The antibody was prepared as described by Schier et al.(Immunotechnology 1:73-81 (1995)). The reaction and products assay wereconducted as described in the Example above. Total protein recovery was86%. Approximately 52% of the recovered protein (M.w. 27,000) was in theform of a product with higher molecular weight (M.w. 29,000-30,000),consistent with the expected conjugate.

Example 6

Preparation of inimunoliposomes with conjugated anti-HER2 Fab′ fragmentsand loaded with a fluorescent ph-sensitive indicator

Small (100 mn) unilamellar liposomes containing entrapped pH-sensitivefluorescent indicator 8-hydroxypyrene trisulfonic acid were preparedfrom a mixture of 1-palmitoyl-2oleoyl-phosphatidylcholine (Avanti),cholesterol (Calbiochem), and methoxypolyoxyethyleneglycol (M.w.1,900)-derivatized distearoyl phosphatidylethanolamine (Sygena) in themolar ratio of 30:20:3 as described by Kirpotin et al. (Biochemistry,36:66-75 (1997)), and sterilized by filtration through 0.2 μm celluloseacetate filter. 0.26 ml of liposome preparation containing 2 μmol ofphospholipids was mixed with 0.106 ml of a solution containing 100 μg ofthe anti-HER2 Fab′-PEG-DSPE conjugate prepared according to Example 4,above, and incubated overnight at 37° C. Following incubation, theliposomes were separated from unbound material by gel-filtration on acolumn with Sepharose 4B (Pharmacia), using HEPES-buffered saline aseluent. The liposomes were eluted in the void volume of the column. Theamount of liposome-bound protein was determined by the Bio-Rad dyebinding assay, and the liposome concentration was measured by totalphosphorus using molybdate method (Morrison, Anal. Biochem., 7:218-224(1964). SDS-PAGE of the liposomes (see Example 13, below) revealed thepresence of anti-HER2 Fab′-PEG-DSPE conjugate, but no free anti-HER2Fab′ in the liposome preparation. Liposome-associated protein wasquantified by SDS-PAGE (see Example 13) and binding of the addedFab′-PEG-DSPE conjugate with the liposomes was expressed as percentageof the output protein/phospholipid ratio over the inputprotein/phospholipid ratio. The binding of Fab′-PEG-DSPE conjugate tothe liposomes was 80%. The leakage of HPTS from the liposomes duringincubation with the protein-PEG-DSPE conjugate to the liposomes was lessthan 2%.

Example 7

Preparation of immunoliposomes with conjugated anti-HER2 scFv antibodiesand loaded with a fluorescent pH-sensitive indicator

Using the procedure of Example 6, the conjugate of anti-HER2 singlechain Fv C6.5Cys with Mal-PEG-DSPE, obtained according to Example 5, wasincubated with HPTS-loaded liposomes at the input ratio of 15.6 μg ofprotein per 1 μmol of liposome phospholipid. After separation of unboundmaterial by gel-filtration on Sepharose 4B, the liposomes were assayedas described in Example 6. The output protein/phospholipid ratio was14.4 μg/ttmol, which indicated 92.3% binding of the conjugate to theliposomes.

Example 8

Uptake of the liposomes by HER2-overexpressing cells.

HER2-overexpressing human breast cancer cells (SK-BR-3) were grown inMcCoy SA medium supplemented with 10% fetal calf serum, 50 U/ml ofpenicillin, and 50 U/ml of streptomycin at 37° C. and 5% CO₂. Twentyfour hours prior to assay, the cells were harvested by treatment with 5mM EDTA in phosphate buffered saline, and plated into 24-well cellculture plates at a density of 200,000 cells/well in 1 ml of cellculture medium. Liposomes were added to the cell culture medium in thewells (in triplicates) to achieve a final concentration of 25 μM ofliposome phospholipids. The plates were then incubated 4 hours withgentle agitation at 37° C. and 5% CO₂. After incubation the media wereaspirated from the wells, the cell layers were rinsed four times with 1ml of phosphate buffered saline, harvested into 1 ml of 5 mM EDTA inphosphate buffered saline, and the amounts of cell-bound and endocytosedliposomes were determined by fluorometry as described in Kirpotin etal., Biochemistry, 36:66-75 (1997). For comparison, incubations werealso performed with the liposomes conjugated to anti-HER2 Fab′ and scFvvia Mal-PEG-DSPE linkers pre-included into the liposome composition(Kirpotin et al., Ibid). The results are summarized in the followingtable:

Total cell-associated Endocytosed Proteins per liposomes, nmolliposomes, Liposomes liposome phospholipid/10⁶ cells % of total Noantibody  0  0.0059 ± 0.00036 0 anti-HER2 Fab′, 34 0.744 ± 0.086  86 ±7.8 conjugation to pre-incorporated linker anti-HER2 scFv, 37 0.311 ±0.025 59.3 ± 4.3  conjugation to pre-incorporated linker anti-HER2 Fab′,43 1.304 ± 0.054 95.9 ± 3.2  according to Example 4 anti-HER2 scFv, 390.576 ± 0.035 60.4 ± 0.9  according to Example 5

As evidenced by these data, target cell binding and internalization ofthe liposomes prepared according to the present invention was at leastequal, and often superior to, that of the similar liposomes preparedaccording to the best prior method.

Example 9

Preparation of anti-HER2 immunoliposomal doxorubicin by modification ofpremanufactured liposomal doxorubicin with anti HER2 Fab′-PEG-DSPEconjugate at 55° C.

0.38 ml of commercially available liposomal doxorubicin (Doxil®, SequusPharmaceuticals, Inc.) containing 2 mg/ml of doxorubicin was mixed with0.26 ml of the preparation of anti HER2 Fab′-PEG-DSPE conjugate obtainedaccording to Example 6, incubated at 55° C. for 20 min., and quicklycooled down in ice-water. Unbound material and low-molecular componentswere removed by gel-filtration of the incubation products through acolumn with Sepharose 4B (Pharmacia). The liposomes were collected inthe void volume of the column, and assayed for protein using SDS-PAGE,for phospholipid using the molybate method, and for doxorubicin byspectrophotometry after solubilization in acidified isopropanol (E^(1%)₄₈₀=208). Found: approx. 45 Fab′/liposome (77% binding of the addedconjugate). The leakage of doxorubicin from liposomes was not observed(doxorubicin content prior to incubation, 145.9 μg/,mol phospholipid;after incubation, 155.8 μg/mol phospholipid).

Example 10

Preparation of anti-HER2 immunoliposomal doxorubicin by modification ofpremanufactured liposomal doxorubicin with anti-HER2 scFv-PEG-DSPEconjugate at 55° C.

The modification was performed as described in Example 9, using 0.4 mlof C6.5Cys-PEG-DSPE conjugate preparation (Example 5) and 0.31 ml ofDoxilu. Found: 48 proteins/liposome (quantitative binding of theconjugate to liposomes); drug leakage 3.7% (doxorubicin content prior tomodification, 145.9 μg/Amol phospholipid; after modification, 140.5μg/μmol phospholipid).

Example 11

Preparation of anti-HER2 immunoliposomal doxorubicin by modification ofDoxil® with anti-HER2 Fab′-PEG-DSPE conjugate at 37° C.

The modification was performed as described in Example 9 above, using0.31 ml of Doxil® and 0.212 ml of anti-HER2 Fab′-PEG-DSPE preparation(Example 4), but the incubation was overnight at 37° C. Found: 46Fab′/liposome (82% binding of the added conjugate to liposomes); drugleakage was not observed (doxorubicin prior to modification 145.9μg/μmol phospholipid; after modification, 146.0 μg/μmol phospholipid).Transition temperature of the lipid constituent of Doxil® (hydrogenatedsoy phosphatidylcholine) is close to 55° C. Thus, modification isequally effective when the liposome lipids are in the gel state.

Example 12

Preparation of anti-HER2 immunoliposomal doxorubicin by modification ofDoxil® with anti-HER2 scFv-PEG-DSPE conjugate at 37° C.

The modification was performed as described in Example 11, above, using0.31 ml of Doxil® and 0.4 ml of C6.5Cys-PEG-DSPE conjugate preparation(Example 5). Found: 49 proteins/liposome (quantitative binding of theconjugate to liposomes). Drug leakage was not detected (doxorubicinprior to modification 145.9 μg/μmol phospholipid; after modification,150.3.0 μg/μmol phosholipid). Thus, modification of the liposomes withscFv-PEG-DSPE conjugate was equally effective when the liposome lipidswere in the gel state.

Example 13

Quantitation of antibody conjugate in the liposomes and conjugationproducts prepared according to Examples 6-12.

The amount of protein-PEG conjugate in the conjugation product and inthe liposomes was assayed by polyacrylamide gel electrophoresis in thepresence of sodium dodecylsulfate (SDS-PAGE) under non-reducingconditions according to Laemmli (1974). Typically, 5-20 μL aliquots ofanalytical sample were mixed with 6× sample buffer containing SDS andtrack dye (bromophenol blue), incubated 1 min. at 60° C., and appliedonto a polyacrylamide gel (dimensions 10×10×0.075 cm) with aconcentration of 10-12%, and cross-linker content of 2.6%. Theseparation was effected in a vertical slab gel electrophoresis apparatusat constant current of 30 mA. The protein bands were developed byCoomassie Blue staining using conventional methods. The conjugate formeda distinct band with lower electrophoretic mobility than the originalprotein. For quantitation of protein, the bands were excised, and thedye was extracted into 50% aqueous dimethylformamide at 100° C. for 30min. The amount of extracted dye was quantified by spectrophotometry at595 nm, and the protein amount per band was determined by comparison toa standard curve produced from the similarly processed bands ofconcomitantly run standard amounts of corresponding protein 9(Fab′ orscFv).

Example 14

Delivery of doxorubicin to HER2-overexpressing cancer cells by anti-HER2immunoliposomes prepared according to Examples 9-12.

HER2-overexpressing human breast cancer cells (SK-BR-3) were grown andplated as described in Example 8, above. Preparations of anti-HER2immunoliposomal doxorubicin (Examples 9-12 above) were added to the cellculture medium in the wells (in triplicates) to achieve final 200 1Mconcentration of liposome phospholipids (0.030±0.001 mg/ml ofdoxorubicin). The plates were then incubated 4 hours with gentleagitation at 37° C. and 5% CO₂. After incubation the liquid wasaspirated from the wells, the cell layers were rinsed 3 times with 1 mleach time of phosphate buffered saline, and the cells were harvestedinto 0.5 ml of 5 mM EDTA in phosphate buffered saline, pelleted bycentrifugation, and extracted with 0.3N HC1/50% ethanol mixture. Theamount of doxorubicin in ethanol-HCI extracts was determined byspectrofluorometry (excitation wavelength, 470 nm; emission wavelength590 nm) and normalized to the quantity of plated cells. For comparison,incubations were performed also with the liposomes conjugated toanti-HER2 scFv (C6.5Cys) via Mal-PEG-DSPE linkers pre-incorporated intothe liposome lipid matrix (Kirpotin et al., 1997). To assess thespecificity of binding, in some wells the cells were preincubated with 5μg of the free anti-HER2 bivalent monoclonal antibody (anti-HER2MAb).The results are summarized in the following table:

Doxorubicin uptake, Liposomal doxorubicin preparation: pg/cell (mean ±SE) Example 9 1.652 ± 0.046 Example 10 1.364 ± 0.016 Example 11 1.518 ±0.040 Example 12 1.118 ± 0.005 anti-HER2 scFv, conjugation to liposome-0.372 ± 0.015 incorporated active linker Example 9 + anti-HER2MAb 0.372± 0.015

Immunoliposomes prepared according to the present invention were capableof delivery of liposome-encapsulated doxorubicin to target cells evenmore efficiently than immunoliposomes prepared by previous methods, i.e.conjugation of the antibody fragment to the liposomes containingactivated linker. Preincubation of the cells with free antibody reactiveto the target antigen (HER2 protein) on the cell surface caused tenfolddecrease in the uptake of immunoliposomal doxorubicin prepared accordingto the present invention; therefore, the uptake was target-specific.

Example 15

Preparation of lipid-DNA complex microparticles with conjugated antibodyfragments

A suspension of lipid-DNA microparticles (measuring 410±150 nm in sizeby dynamic laser scattering) composed of plasmid DNA (pCMV/IVS-Luc⁺; 10μg/mL), dimethyl dioctadecylammonium bromide (DDAB, 60 nmol/mL), anddioleoyl phosphatidylethanolarmine (DOPE, 60 mnol/mL) in 5% aqueousdextrose, was prepared as described by Hong et al. (FEBS lett.400:233-237, 1997). Fab′-PEG-DSPE conjugate was prepared byco-incubation of Mal-PEG-DSPE and anti-HER2 antibody Fab′ fragments at amolar ratio of 4:1, at a concentration of the protein of 0.3 mg/mL inaqueous physiological buffer, at pH 7.2 for 2 hours. Lipid-DNAmicroparticles with conjugated anti-HER2 Fab′ fragments were prepared byincubation of the lipid-DNA microparticles with the conjugate in theamount of 0.5 mol. % relative to total particle lipid content for atleast 30 min. at room temperature. Control particles with linker alone(non-targeted control) were prepared in the similar manner, butnon-conjugated, β-mercaptoethanol-quenched Mal-PEG-DSPE was substitutedfor the Fab′-PEG-DSPE conjugate.

Example 16

Targeted DNA transfection of the cells by lipid-DNA microparticles withconjugated antibody fragments

Transfection activity of pCMV/IVS-Luc⁺ DNA-lipid microparticles preparedas in Example 15, above was studied in the cultures of human breastcancer cells: SK-BR-3 (overexpressing the target antigen, HER2oncoprotein) and MCF-7 (the line with low expression of HER2).Expression of the reporter gene (luciferase) was determined byluminometry after 24-hour exposure of the cells to lipid-DNA complexes(1 μg of DNA per 50-100,000 cells) in 10% serum-supplemented growthmedium, and served as the measure of transfection efficiency. Thedetailed description of this experimental procedure is given in Hong etal., FEBS Lett. 400:233-237 (1997). Anti-HER2 Fab′ conjugated DNA-lipidmicroparticles prepared according to this invention were about 25-timesmore efficient for the plasmid delivery to target-positive SK-BR-3 cellsthan matching non-targeted particles. In the target-negative MCF-7cells, targeted and nontargeted DNA-lipid particles had equalefficiency. Thus, antibody-modified lipid-DNA particles preparedaccording to the invention, are capable of target-specific delivery offunctional DNA into human cancer cells.

Luciferase expression, ng/mg cell protein Cells: Microparticles (mean ±SE) SK-BR-3 DNA/lipid alone 116.2 ± 35.4  SK-BR-3 DNA/lipid + Mal-PEG-40.4 ± 0.1  DSPE (*non-target control) SK-BR-3 DNA-lipid + Fab′-PEG- 995± 197 DSPE (targeted) MCF-7 DNA/lipid alone 6.44 ± 0.34 MCF-7DNA/lipid + Mal-PEG- 0.58 ± 0.30 DSPE (*non-target control) MCF-7DNA-lipid + Fab′-PEG- 0.71 ± 13   DSPE (targeted)

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

What is claimed is:
 1. A method for preparing a lipidic microparticleattached to a protein by means of a linker molecule, said methodcomprising the step of: incubating a lipidic microparticle with aprotein conjugated to a linker molecule comprising a hydrophobic domain,a hydrophilic polymer chain terminally attached to the hydrophobicdomain, and a chemical group reactive to one or more functional groupson a protein molecule and attached to the hydrophilic polymer chain at aterminus contralateral to the hydrophobic domain, for a time sufficientto permit the hydrophobic domain to become stably associated with thelipidic microparticle.
 2. A method for preparing a lipidic microparticleattached to a protein, said method comprising the step of: incubating aprotein comprising a terminally appended amino acid sequence comprisingprimarily amino acids with hydrophilic side chains, which sequence isfollowed by a lipid modification site with a synthetically appendedlipid moiety, with a lipidic microparticle for a time sufficient topermit the lipid moiety to become stably associated with the lipidicmicroparticle.
 3. The method of claim 1, wherein the lipidicmicroparticle is a liposome.
 4. The method of claim 1, wherein thelipidic microparticle is a lipid:nucleic acid complex.
 5. The method ofclaim 1, wherein the lipidic microparticle is a lipid:drug complex. 6.The method of claim 1, wherein the lipidic microparticle is amicroemulsion droplet.
 7. The method of claim 1, wherein the protein isan antibody.
 8. The method of claim 1, wherein the protein is an Fab′fragment of an antibody.
 9. The method of claim 1, wherein the proteinis single chain Fv antibody.
 10. The method of claim 1, wherein theprotein is an enzyme.
 11. The method of claim 1, wherein the protein isa hormone.
 12. The method of claim 1, wherein the protein is a growthfactor.
 13. The method of claim 1, wherein the protein is a nucleic acidbinding protein.
 14. The method of claim 1, wherein the reactive groupis a maleimido group.
 15. The method of claim 1, wherein the incubationoccurs in an aqueous medium.
 16. The method of claim 1, wherein theconjugated protein undergoes a purification step to separate it fromunreacted linker and unmodified protein prior to incubation.
 17. Themethod of claim 16, wherein the purification step is selected from thegroup consisting of salting-out, dialysis, and chromatography.
 18. Alipidic microparticle conjugated to a protein by the method of claim 1,wherein the protein is conjugated to the lipidic microparticle through alinker molecule by means of a chemical group, which chemical group priorto conjugation is reactive with a component of said lipidicmicroparticle.
 19. A liposome conjugated to a protein by the method ofclaim 1, wherein the protein is conjugated to the liposome through alinker molecule by means of a chemical group, which chemical group priorto conjugation is reactive with a component of said liposome.
 20. Alipid:nucleic acid complex conjugated to a protein by the method ofclaim
 1. 21. A lipid:drug complex conjugated to a protein by the methodof claim 1, wherein the protein is conjugated to the lipid:drug complexthrough a linker molecule by means of a chemical group, which chemicalgroup prior to conjugation is reactive with a component of saidlipid:drug complex.
 22. A microemulsion droplet conjugated to a proteinby the method of claim 1, wherein the protein is conjugated to themicroemulsion droplet through a linker molecule by means of a chemicalgroup, which chemical group prior to conjugation is reactive with acomponent of said microemulsion droplet.
 23. A lipid:nucleic acidcomplex conjugated to a protein by the method of claim 1, wherein theprotein is conjugated to the lipid:nucleic acid complex through a linkermolecule by means of a chemical group, which chemical group prior toconjugation is reactive with a component of said lipid:nucleic acidcomplex.
 24. A lipidic microparticle conjugated to a protein, whereinthe protein is conjugated to the lipidic microparticle through a linkermolecule comprising a hydrophobic domain, a hydrophilic polymer chainterminally attached to the hydrophobic domain, and a chemical groupreactive to one or more functional groups on a protein molecule andattached to the hydrophilic polymer chain at a terminus contralateral tothe hydrophobic domain, wherein the chemical group prior to conjugationis reactive with a component of said lipidic microparticle.
 25. Alipidic microparticle of claim 24, wherein the lipidic microparticle isselected from the group consisting of: a liposome, a lipid:nucleic acidcomplex, a lipid:drug complex, and a microemulsion droplet.
 26. Alipidic microparticle conjugated to a protein by the method of claim 1,wherein the microparticle does not bear unconjugated linkers.
 27. Aliposome conjugated to a protein by the method of claim 1, wherein themicroparticle does not bear unconjugated linkers.
 28. A lipid:nucleicacid complex conjugated to a protein by the method of claim 1, whereinthe microparticle does not bear unconjugated linkers.
 29. A lipid:drugcomplex conjugated to a protein by the method of claim 1, wherein themicroparticle does not bear unconjugated linkers.
 30. A microemulsiondroplet conjugated to a protein by the method of claim 1, wherein themicroparticle does not bear unconjugated linkers.
 31. A lipidicmicroparticle conjugated to a protein through a linker moleculecomprising a hydrophobic domain, a hydrophilic polymer chain terminallyattached to the hydrophobic domain, and a chemical group reactive to oneor more functional groups on a protein molecule and attached to thehydrophilic polymer chain at a terminus contralateral to the hydrophobicdomain, wherein the lipidic microparticle does not bear unconjugatedlinkers.
 32. A lipidic microparticle of claim 31, wherein the lipidicmicroparticle is selected from the group consisting of: a liposome, alipid:nucleic acid complex, a lipid:drug complex, and a microemulsiondroplet.
 33. A microparticle which is conjugated to two or more proteinspecies by the method of claim 1, wherein the protein species areconjugated to the microparticle through a functional group, whichfunctional group is the same for each protein species.
 34. A liposomewhich is conjugated to two or more protein species by the method ofclaim 1, wherein the protein species are conjugated to the liposomethrough a functional group, which functional group is the same for eachprotein species.
 35. A lipid:nucleic acid complex which is conjugated totwo or more protein species by the method of claim 1, wherein theprotein species are conjugated to the lipid:nucleic acid complex througha functional group, which functional group is the same for each proteinspecies.
 36. A lipid:drug complex which is conjugated to two or moreprotein species by the method of claim 1, wherein the proteins areconjugated to the lipid:drug complex through a functional group, whichfunctional group is the same for each protein species.
 37. Amicroemulsion droplet which is conjugated to two or more protein speciesby the method of claim 1, wherein the proteins are conjugated to themicroemulsion droplet through a functional group, which functional groupis the same for each protein species.
 38. A lipidic microparticle whichis conjugated to two or more protein species, wherein the proteinspecies are conjugated to the lipidic microparticle through linkermolecules, each linker molecule comprising a hydrophobic domain, ahydrophilic polymer chain terminally attached to the hydrophobic domain,and a chemical group reactive to one or more functional groups on saidprotein molecule conjugated to said linker, and attached to thehydrophilic polymer chain at a terminus contralateral to the hydrophobicdomain, which functional groups are the same for each protein species.39. A lipidic microparticle of claim 38, wherein the lipidicmicroparticle is selected from the group consisting of: a liposome, alipid:nucleic acid complex, a lipid:drug complex, and a microemulsiondroplet.
 40. A method of claim 1, wherein two or more protein speciesare attached to said lipidic microparticle.
 41. A method of claim 40,wherein said protein species are independently selected from the groupconsisting of: an antibody, an Fab′, and a single-chain Fv antibody. 42.A method of claim 40, wherein said lipidic microparticle is a liposome.43. A method for attaching a plurality of protein species to a lipidicmicroparticle, wherein at least one of the protein species is attachedby the method of claim
 1. 44. A method of claim 1, wherein the chemicalgroup reactive to one or more functional groups on said protein isreactive to a component of the lipidic microparticle.
 45. A method ofclaim 44, wherein the lipidic microparticle is selected from the groupconsisting of: a liposome, a lipid:nucleic acid complex, a lipid:drugcomplex, and a microemulsion droplet.
 46. A method of claim 44, whereinthe chemical group is selected from the group consisting of: an aminogroup, a carboxy group, a thiol group, a malemido group, aniodoacetamido group, a vinylsulfone group, an aldehyde group, ahydrazine group, a ketone group, and a cyanure chloride group.